Current Developments in Biotechnology and Bioengineering. Production, Isolation and Purification of Industrial Products 1st Edition Ashok Pandey

Download More ebooks [PDF]. Format PDF ebook download PDF KINDLE.
Full download ebooks at ebookmass.com
Current Developments in Biotechnology

and Bioengineering. Production,
Isolation and Purification of Industrial
Products 1st Edition Ashok Pandey
For dowload this book click BUTTON or LINK below
https://ebookmass.com/product/current-
developments-in-biotechnology-and-bioengineering-
production-isolation-and-purification-of-
industrial-products-1st-edition-ashok-pandey/
OR CLICK BUTTON
DOWLOAD NOW
Download More ebooks from https://ebookmass.com

More products digital (pdf, epub, mobi) instant
download maybe you interests ...
Current Developments in Biotechnology and

Bioengineering. Food and Beverages Industry 1st Edition
Ashok Pandey
https://ebookmass.com/product/current-developments-in-
biotechnology-and-bioengineering-food-and-beverages-industry-1st-
edition-ashok-pandey/

Bioengineering: Advances in Composting and
Vermicomposting Technology Ashok Pandey
biotechnology-and-bioengineering-advances-in-composting-and-
vermicomposting-technology-ashok-pandey/

Bioengineering. Biological Treatment of Industrial
Effluents 1st Edition Duu-Jong Lee
biotechnology-and-bioengineering-biological-treatment-of-
industrial-effluents-1st-edition-duu-jong-lee/

Bioengineering: Bioremediation of Endocrine Disrupting
Pollutants in Industrial Izharul Haq
biotechnology-and-bioengineering-bioremediation-of-endocrine-
disrupting-pollutants-in-industrial-izharul-haq/
Bioengineering: Filamentous Fungi Biorefinery Mohammad
Taherzadeh
biotechnology-and-bioengineering-filamentous-fungi-biorefinery-
mohammad-taherzadeh/

Bioengineering : Designer Microbial Cell Factories
Swati Joshi
biotechnology-and-bioengineering-designer-microbial-cell-
factories-swati-joshi/

Bioengineering. Solid Waste Management 1st Edition
Jonathan W-C Wong
biotechnology-and-bioengineering-solid-waste-management-1st-
edition-jonathan-w-c-wong/

Bioengineering. Human and Animal Health Applications
1st Edition Vanete Thomaz Soccol
biotechnology-and-bioengineering-human-and-animal-health-
applications-1st-edition-vanete-thomaz-soccol/

Bioengineering: Biochar Towards Sustainable Environment
Huu Hao Ngo
biotechnology-and-bioengineering-biochar-towards-sustainable-
environment-huu-hao-ngo/
Current Developments
in Biotechnology and
Bioengineering
Production, Isolation and Purification
of Industrial Products
Edited by
Ashok Pandey, Sangeeta Negi,
Carlos Ricardo Soccol
AMSTERDAM l BOSTON l HEIDELBERG l LONDON l NEW YORK l OXFORD

PARIS l SAN DIEGO l SAN FRANCISCO l SINGAPORE l SYDNEY l TOKYO
Elsevier
Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands
The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom
50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States
Copyright © 2017 Elsevier B.V. All rights reserved.

No part of this publication may be reproduced or transmitted in any form or by any means,
electronic or mechanical, including photocopying, recording, or any information storage
and retrieval system, without permission in writing from the publisher. Details on how to
seek permission, further information about the Publisher’s permissions policies and our
arrangements with organizations such as the Copyright Clearance Center and the Copyright
Licensing Agency, can be found at our website: www.elsevier.com/permissions.
This book and the individual contributions contained in it are protected under copyright by
the Publisher (other than as may be noted herein).
Notices
Knowledge and best practice in this field are constantly changing. As new research and
experience broaden our understanding, changes in research methods, professional practices,
or medical treatment may become necessary.
Practitioners and researchers must always rely on their own experience and knowledge in
evaluating and using any information, methods, compounds, or experiments described herein.
In using such information or methods they should be mindful of their own safety and the safety
of others, including parties for whom they have a professional responsibility.
To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors,
assume any liability for any injury and/or damage to persons or property as a matter of
products liability, negligence or otherwise, or from any use or operation of any methods,
products, instructions, or ideas contained in the material herein.
Library of Congress Cataloging-in-Publication Data
A catalog record for this book is available from the Library of Congress
British Library Cataloguing-in-Publication Data

A catalogue record for this book is available from the British Library
ISBN: 978-0-444-63662-1
For information on all Elsevier publications

visit our website at https://www.elsevier.com/
Publisher: John Fedor

Acquisition Editor: Kostas Marinakis
Editorial Project Manager: Anneka Hess
Production Project Manager: Vijayaraj Purushothaman
Designer: Greg Harris
Typeset by TNQ Books and Journals

List of Contributors
M. Adsul DBT-IOC Centre for Advanced Bioenergy Research, IndianOil Corporation

Limited
Cristóbal N. Aguilar Food Research Department, School of Chemistry,

Autonomous University of Coahuila, Saltillo, Coahuila, México
A. Angel-Cuapio Universidad Autónoma Metropolitana-Iztapalapa, Mexico City,

DF, Mexico
G.S. Anisha Government College for Women, Trivandrum, Kerala, India
P. Binod CSIR-National Institute for Interdisciplinary Science and Technology (NIIST),

Trivandrum, India
J. Buenrostro-Figueroa Department of Biotechnology, Division of Health and

Biological Sciences, Metropolitan Autonomous University, Iztapalapa, México
S. Chakraborty Indian Institute of Technology Guwahati, Guwahati, Assam, India
M.L. Chávez González Food Research Department, School of Chemistry,

G.-Q. Chen Tsinghua University, Beijing, China
S. Chen Hubei University, Wuhan, PR China
Juan C. Contreras-Esquivel Food Research Department, School of Chemistry,

J.D. Coral Bioprocess Engineering and Biotechnology Department, Federal

University of Paraná (UFPR), Curitiba, PR, Brazil
J.C. de Carvalho Bioprocess Engineering and Biotechnology Department, Federal

xxi
xxii List of Contributors
J. de Oliveira Bioprocess Engineering and Biotechnology Department, Federal

A. Dhillon Indian Institute of Technology Guwahati, Guwahati, Assam, India
M.J. Fernandes Bioprocess Engineering and Biotechnology Department, Federal

R. Gaur Indian Oil Corporation Limited, R&D Centre, Faridabad, India
A. Goyal Indian Institute of Technology Guwahati, Guwahati, Assam, India
L.R.C. Guimarães Bioprocess Engineering and Biotechnology Department, Federal

M. Haridas Kannur University, Kannur, India
R. Hemamalini Indian Institute of Technology Delhi, New Delhi, India
Ayerim Hernandez-Almanza Food Research Department, School of Chemistry,

A. Illanes Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile
J. Isar University of Delhi South Campus, New Delhi, India
A. Joseph Kannur University, Kannur, India
S.G. Karp Bioprocess Engineering and Biotechnology Department, Federal

N. Karthik CSIR-National Institute for Interdisciplinary Science and Technology

(NIIST), Trivandrum, India
R. Kaushik University of Delhi South Campus, New Delhi, India
S.K. Khare Indian Institute of Technology Delhi, New Delhi, India
P.C.S. Kirnev Bioprocess Engineering and Biotechnology Department, Federal

D. Kothari Indian Institute of Technology Guwahati, Guwahati, Assam, India

List of Contributors xxiii
C. Larroche Blaise Pascal University, Aubière Cedex, France
L.A.J. Letti Bioprocess Engineering and Biotechnology Department, Federal

O. Loera-Corral Universidad Autónoma Metropolitana-Iztapalapa, Mexico City, DF,

Mexico
A.I. Magalhães, Jr. Bioprocess Engineering and Biotechnology Department,

Federal University of Paraná (UFPR), Curitiba, PR, Brazil
A.B.P. Medeiros Bioprocess Engineering and Biotechnology Department, Federal

J.D.C. Medina Bioprocess Engineering and Biotechnology Department, Federal

F. Miranda-Hernández Universidad Autónoma Metropolitana-Iztapalapa, Mexico

City, DF, Mexico
N.R. Nair CSIR-National Institute for Interdisciplinary Science and Technology

S. Nair Dow Chemicals GmBH, Dubai, UAE
K.M. Nampoothiri CSIR-National Institute for Interdisciplinary Science and

Technology (NIIST), Trivandrum, India
A. Nandan CSIR-National Institute for Interdisciplinary Science and Technology

S. Negi Motilal Nehru National Institute of Technology, Allahabad, India
M.G.B. Pagnoncelli Bioprocess Engineering and Biotechnology Department,

Federal University of Paraná (UFPR), Curitiba, PR, Brazil; Federal Technological
University of Parana, Dois Vizinhos, Brazil
A. Pandey Center of Innovative and Applied Bioprocessing, (a national institute

under Dept of Biotechnology, Ministry of S&T, Govt of India), Mohali, Punjab, India
A.K. Patel DBT-IOC Centre for Advanced Bioenergy Research, IndianOil Corporation
Limited
xxiv List of Contributors
V. Rajulapati Indian Institute of Technology Guwahati, Guwahati, Assam, India
S. Ramachandran Insight Professional Institute, Dubai, UAE
A. Rani Indian Institute of Technology Guwahati, Guwahati, Assam, India
C. Rodrigues Bioprocess Engineering and Biotechnology Department, Federal

Rosa M. Rodríguez-Jasso Food Research Department, School of Chemistry,

R. Rodríguez Food Research Department, School of Chemistry, Autonomous

University of Coahuila, Saltillo, Coahuila, México
L.V. Rodríguez Durán Department of Biotechnology, Division of Health and

Biological Sciences, Metropolitan Autonomous University, Iztapalapa, México
Héctor A. Ruiz Food Research Department, School of Chemistry, Autonomous

A. Sabu Kannur University, Kannur, India
R. Saini DBT-IOC Centre for Advanced Bioenergy Research, IndianOil Corporation

Limited
S. Sajitha CSIR-National Institute for Interdisciplinary Science and Technology

S. Saran University of Delhi South Campus, New Delhi, India
R.K. Saxena University of Delhi South Campus, New Delhi, India
V.C. Sekhar CSIR-National Institute for Interdisciplinary Science and Technology

K. Sharma Indian Institute of Technology Guwahati, Guwahati, Assam, India
R. Sindhu CSIR-National Institute for Interdisciplinary Science and Technology

R.P. Singh Punjabi University, Patiala, Punjab, India

List of Contributors xxv
R.S. Singh Punjabi University, Patiala, Punjab, India
Reeta R. Singhania DBT-IOC Centre for Advanced Bioenergy Research, IndianOil

Corporation Limited
C.R. Soccol Bioprocess Engineering and Biotechnology Department, Federal

T.S. Swapna Government Victoria College, Palakkad, India
D. Tan Xían Jiaotong University, Xían, China
L. Thomas CSIR-National Institute for Interdisciplinary Science and Technology

M.V. Ushasree CSIR-National Institute for Interdisciplinary Science and Technology

P. Valencia Universidad Técnica Federico Santa María, Valparaíso, Chile
L.P.S. Vandenberghe Bioprocess Engineering and Biotechnology Department,

K. Vibha Motilal Nehru National Institute of Technology, Allahabad, India
J. Vidya CSIR-National Institute for Interdisciplinary Science and Technology (NIIST),

Trivandrum, India
N. Vijayan Kannur University, Kannur, India
N. Vivek CSIR-National Institute for Interdisciplinary Science and Technology (NIIST),

Trivandrum, India
Q. Wang Hubei University, Wuhan, PR China
X. Wei Hubei University, Wuhan, PR China
A.L. Woiciechowski Bioprocess Engineering and Biotechnology Department,

J. Yin Tsinghua University, Beijing, China

xxvi List of Contributors
A. Zandoná Filho Bioprocess Engineering and Biotechnology Department, Federal

P.A. Zárate Food Research Department, School of Chemistry, Autonomous

S.F. Zawadzki Bioprocess Engineering and Biotechnology Department, Federal

About the Editors
Ashok Pandey
Professor Ashok Pandey is Eminent Scientist at the Center of
Innovative and Applied Bioprocessing, Mohali (a national
institute under the Department of Biotechnology, Ministry
of Science and Technology, Government of India), and
former chief scientist and head of the Biotechnology
Division at the CSIR’s National Institute for Interdisciplinary
Science and Technology at Trivandrum. He is an adjunct
professor at Mar Athanasios College for Advanced Studies
Thiruvalla, Kerala, and at Kalasalingam University, Krishnan
Koil, Tamil Nadu. His major research interests are in the
areas of microbial, enzyme, and bioprocess technology,
which span various programs, including biomass to fuels
and chemicals, probiotics and nutraceuticals, industrial
enzymes, solid-state fermentation, etc. He has more than
1100 publications and communications, which include 16
patents, 50+ books, 125 book chapters, and 425 original and review papers, with an h index
of 75 and more than 23,500 citations (Google Scholar). He has transferred several tech-
nologies to industries and has been an industrial consultant for about a dozen projects for
Indian and international industries.
Professor Pandey is the recipient of many national and international awards
and fellowships, which include Elected Member of the European Academy of Sciences
and Arts, Germany; Fellow of the International Society for Energy, Environment and
Sustainability; Fellow of the National Academy of Science (India); Fellow of the Biotech
Research Society, India; Fellow of the International Organization of Biotechnology and
Bioengineering; Fellow of the Association of Microbiologists of India; honorary doctorate
degree from the Université Blaise Pascal, France; Thomson Scientific India Citation
Laureate Award, United States; Lupin Visiting Fellowship; Visiting Professor at the
Université Blaise Pascal, France, the Federal University of Parana, Brazil, and the École
Polytechnique Fédérale de Lausanne, Switzerland; Best Scientific Work Achievement
Award, Government of Cuba; UNESCO Professor; Raman Research Fellowship Award,
CSIR; GBF, Germany, and CNRS, France fellowships; Young Scientist Award; and others.
He was chairman of the International Society of Food, Agriculture and Environment,
Finland (Food & Health) during 2003e04. He is the Founder President of the Biotech
xxvii
xxviii About the Editors
Research Society, India (www.brsi.in); International Coordinator of the International

Forum on Industrial Bioprocesses, France (www.ifibiop.org); chairman of the
International Society for Energy, Environment & Sustainability (www.isees.org); and vice
president of the All India Biotech Association (www.aibaonline.com). Professor Pandey
is editor-in-chief of Bioresource Technology, Honorary Executive Advisor of the Journal of
Water Sustainability and Journal of Energy and Environmental Sustainability, subject
editor of the Proceedings of the National Academy of Sciences (India), and editorial board
member of several international and Indian journals, and also a member of several
national and international committees.
Sangeeta Negi
Dr. Sangeeta Negi is an assistant professor in the Department
of Biotechnology at the Motilal Nehru National Institute of
Technology, India. She has a First Class Master’s degree in
biochemistry and a PhD in biotechnology from the Indian
Institute of Technology, Kharagpur. She has also worked as
an academic guest at the Biological Engineering Department,
Polytech Clermont-Ferrand; the Université Blaise Pascal,
France; and the Bioenergy and Energy Planning Research
Group, Swiss Federal Institute of Technology, Lausanne,
Switzerland. Dr. Negi’s current research interests are in the
areas of biofuels, industrial enzymes, and bioremediation. She is an editorial board
member of the Journal of Waste Conversion, Bioproducts and Biotechnology and the Journal
of Environmental Science and Sustainability. She has been awarded as Outstanding
Reviewer by Elsevier and has won the Young Scientist Award by DST at the National
Seminar on Biological and Alternative Energies Present and Future, organized by Andhra
University, Visakhapatnam, in 2009. She has also won the Best Poster Award at the
International Congress on Bioprocesses in Food Industries (2008) at Hyderabad. Dr. Negi
has contributed to nearly 70 publications, including review articles, original papers, and
conference communications.
About the Editors xxix

Professor Carlos Ricardo Soccol is the research group leader
of the Department of Bioprocesses Engineering and
Biotechnology at the Federal University of Paraná (UFPR),
Brazil, with 20 years of experience in biotechnological
research and development of bioprocesses with industrial
application. He graduated with a BSc in chemical engi-
neering (UFPR, 1979), Master’s in food technology (UFPR,
1986), and PhD in Génie Enzymatique, Microbiologie et
Bioconversion (Université de Technologie de Compiègne,
France, 1992). He did his postdoctoral work at the Institut
ORSTOM/IRD (Montpellier, 1994 and 1997) and at the
Université de Provence et de la Méditerranée (Marseille,
2000). He is an HDR Professor at the École d’Ingénieurs Supériure of Luminy,
MarseilleeFrance. He has experience in the areas of science and food technology, with
emphasis on agro-industrial and agro-alimentary biotechnology, acting in the following
areas: bioprocess engineering and solid-state fermentation, submerged fermentation,
bioseparations, industrial bioprocesses, enzyme technology, tissue culture, bio-
industrial projects, and bio-production. He is currently the Coordinator of Master
BIODEV-UNESCO, associate editor of five international journals, and editor-in-chief of
the journal Brazilian Archives of Biology and Technology. Professor Soccol has received
several national and international awards, which include the Science and Technology
Award of the Government of Paraná (1996); Scopus/Elsevier Award (2009); Dr. Honoris
Causa, Université Blaise Pascal, France (2010); Outstanding Scientist at the 5th
International Conference on Industrial Bioprocesses, Taipei, Taiwan (2012); and Elected
Titular Member of the Brazilian Academy of Sciences (2014). He is a technical and sci-
entific consultant for several companies, agencies, and scientific journals in Brazil and
abroad. He has supervised and mentored 96 Master of Science students, 48 PhD stu-
dents, and 14 postdoctoral students. He has 995 publications and communications,
which include 17 books, 107 book chapters, 270 original research papers, and 557
research communications in international and national conferences and has registered
44 patents. His research articles as of this writing have been cited (Scopus database) 5600
times with an h index of 36.
Preface
This book is a part of the comprehensive series Current Developments in Biotechnology and
Bioengineering, comprising nine volumes (Editor-in-chief: Ashok Pandey), and deals with the
production, isolation, and purification of industrial products produced by biotechnological
processes. This book covers recent technological advances of a great number of biotechno-
logical products and is divided into four different parts: Production of Industrial and
Therapeutic Enzymes, Organic Acids, Biopolymers and Other Products, and Products
Isolation and Purification.
Part 1 is devoted to the production of industrial and therapeutic enzymes. The first
chapter describes the current and future trends of production, application, and strain
improvement of a-amylases, one of the most important enzymes used in industry.
a-Amylases find application in several industrial processes, such as starch liquefaction,
desizing of textiles, detergents, baking, bioethanol production, etc. Glucoamylase is another
enzyme extensively used in the food and fermentation industries, mainly for the saccharifi-
cation of starch, brewing, and production of high-fructose syrup, which are discussed in
Chapter 2. Cellulases, b-glucosidases, and xylanases are the second most used enzymes in
industry by sales volume, with an increasing demand since 1995 in several industrial appli-
cations, comprising detergents and textiles, animal feed, food, paper, and biofuels. These
enzymes are discussed in Chapters 4, 5, and 6 of this book. Chapter 7 discusses proteolytic
enzymes, also known as “proteases,” which are used to cleave the peptide bonds connecting
two amino acids. They are produced mainly by microorganisms and have great commercial
value, being used in food, dairy, detergents, and leather processing. Lipolytic enzymes are
hydrolases comprising 15 families of lipases, as shown in Chapter 8 of this book through a
study of the industrial applications and other important aspects of these enzymes. The
purpose of Chapters 9 and 10 is to present an overview of laccases and peroxidases, covering
their production and use in the pretreatment of lignocellulosic biomass and biopulping, and
also projecting new perspectives on improving such processes and products using these
enzymes. Sources of production, strategies, characteristics, applications, and industrial
importance of therapeutic enzymes, such as L-glutaminase, L-asparaginase, and penicillin
acylase, are presented and discussed in Chapters 11, 12, and 13. Other enzymes, such as
phytases, chitinases, keratinases, tannases, aminopeptidases, nattokinases, and poly-
saccharide lyases, are reviewed in Chapters 14 to 23, covering recent advances, production
methods, potential applications, and the global market.
The second part of the book is dedicated to organic acids. In Chapters 24 and 25, lactic
acid and citric acid production, synthesis (covering factors that affect biochemical pathways),
and recovery are addressed. Chapter 26 reviews the microbial production of gluconic acid,
properties of glucose oxidase, production, recovery, and applications. Succinic acid is an
important platform molecule, used as an intermediate in the production of numerous
everyday products, among which are pharmaceuticals and adhesives, representing a total
immediate addressable market of more than $7.2 billion. Chapter 27 presents an analysis of
the current market, biological-based production processes, enzymatic regulation, and
recovery systems of succinic acid.
xxxi
xxxii Preface
Part 3 discusses polymer production and other products. Polylactide (PLA), derived
from lactic acid, a biodegradable polyester, has applications in packaging, textiles, and the
biomedical and pharmaceutical industries. Chapter 28 reviews the properties and applica-
tions of PLA, focusing on recent technologies and improvement of production techniques.
Polyhydroxyalkanoates (PHAs), a family of environmentally friendly polyesters that can be
synthesized by a wide range of microorganisms as carbon and energy reserves, have been
considered an alternative to petroleum-based chemicals. The composition and structural
diversity of PHAs have led to various properties and endless applications to form a PHA value
chain. Chapter 29 briefly introduces their production and application, highlighting the lab-
oratory production by the microbial strains developed using genetic and/or metabolic en-
gineering or synthetic biology techniques. Industrial production, recent technologies, and
improvement of PHA production are also discussed. Poly-g-glutamic acid (g-PGA) is a natural
polymer, synthesized by various strains of Bacillus spp., that is used in food, cosmetics,
agriculture, and the wastewater industry. Chapter 30 provides updated information on the
biosynthesis, fermentation, purification, and application of g-PGA. In Chapter 31, recent
developments in the biological production of 1,3-propanediol by various natural and
genetically engineered microorganisms, nonnative 1,3-propanediol producers, as well as
mixed cultures, are discussed. Important aspects of downstream processing and various
methods and steps involved in the extraction and purification of 1,3-propanediol from the
fermentation broth are also covered in this chapter. The production of petroleum-based
plastics is a challenging environmental problem, causing the production and consumption
of biodegradable plastics to receive considerable attention nowadays. Chapter 32 provides an
overview of the degradation mechanisms of biodegradable polymers, with particular
emphasis on the main parameters affecting the degradation of these polymeric biomaterials.
In Chapter 33 the potential of biological control is presented and discussed as a promising
alternative to chemical pesticides. The final two chapters of this book, Chapters 34 and 35,
present the most relevant downstream processes to extract, isolate, purify, and refine
fermentation products.
We are confident that this book will be profitable to students, professors, researchers,
and professionals interested in studying biotechnology and bioengineering. We thank
Dr. Kostas Marinakis, Book Acquisition Editor; Ms. Anneka Hess; and entire production team
at Elsevier for their help and support in bringing out this volume.
Editors
Ashok Pandey
Sangeeta Negi
1
a-Amylases
R. Sindhu1, *, P. Binod1, A. Pandey2

1
CSIR-NATIONAL INSTITUTE F OR INTERDISCIPLINARY SCIENCE AND TECHNOLOGY (NIIST),
TRIVANDRUM, INDIA; 2 CE NTER OF INNOVATIVE AND APPLIED BIOPROCESSING,
(A NATIONAL INSTITUTE UNDER DEPT OF BIOTECHNOL OGY, MINISTRY OF S&T, GOVT OF
INDIA), MOHALI, PUNJAB , INDIA
1.1 Introduction
1.1.1 Starch
Starch is the major polysaccharide food reserve in nature after cellulose. It serves as an
important source of nutrition for other living organisms [1]. It is synthesized in the
plastids present in leaves and accumulates as insoluble granules in higher and lower
plants. Starch is composed of a large number of glucose units joined by glycosidic bonds.
It consists of two types of molecules: amylose and amylopectin. Amylose is a linear,
water-insoluble polymer of glucose joined by a-1,4 bonds, whereas amylopectin is a
branched, water-soluble polysaccharide with short a-1,4-linked linear chains of 10e60
glucose units and a-1,6-linked side chains with 15e45 glucose units. The levels of
amylase and amylopectin vary among different starches. Generally, starch is composed
of amylose and amylopectin in the range 25e28% and 72e75%, respectively.
1.1.2 Amylases
Amylases are the enzymes that break down starch, or glycogen. These enzymes are
produced by a variety of living organisms, ranging from bacteria to plants to humans.
Though amylases are produced by several microorganisms, those produced by fungi and
bacteria have dominated applications in the industrial sector [2]. Bacteria and fungi
secrete amylases to the outside of their cells to carry out extracellular digestion, which
breaks down the insoluble starch, and then the soluble end products (such as glucose or
maltose) are absorbed into the cells.
Amylases constitute a class of industrial enzymes occupying about 25% of the enzyme
market. Because of the increasing demand for these enzymes in various industries, there
is enormous interest in developing them with better properties, such as raw starch-
degrading amylases suitable for industrial applications, and cost-effective production
*
Corresponding Author.
Current Developments in Biotechnology and Bioengineering: Production, Isolation and Purification of Industrial Products
http://dx.doi.org/10.1016/B978-0-444-63662-1.00001-4 3
4 CURRENT DEVELOPMENTS IN BIOTECHNOLOGY AND BIOENGINEERING
techniques. Although amylases can be derived from several sources, including plants,
animals, and microorganisms, microbial enzymes generally meet industrial demands.
A large number of microbial amylases are available commercially and they have almost
completely replaced the chemical hydrolysis of starch in the starch processing industry
[3]. One of the most important advantages of using microbes for the production of
amylases is the bulk production capacity and the fact that microbes can be genetically
modified to produce enzymes with desired characteristics [4]. These enzymes are of great
significance in biotechnology, with various applications ranging from food, fermentation,
and textiles to the paper industry. Each application of a-amylase requires unique
properties with respect to specificity, stability, and temperature and pH dependence.
Modern technologies such as computational packages and online servers are the
current tools used in protein sequence analysis and characterization. The physico-
chemical and structural properties of these proteins are well understood with the use of
computational tools. The protein sequence profile, such as number of amino acids and
sequence length, and the physicochemical properties of the protein, such as molecular
weight, atomic composition, extinction coefficient, aliphatic index, instability index, etc.,
can be computed by ProtParam, and the secondary structure prediction, sequence
similarity, evolutionary relationships, and 3-D structure of various proteins can be
computed using the ESyPred3D server [5].
1.1.3 Classification of Amylases

Based on the mechanism of breakdown of starch, the molecules are classified into three
types: a-amylase, b-amylase, and amyloglucosidase. a-Amylase reduces the viscosity of
starch by breaking down the bonds at random, thereby producing variably sized chains
of glucose. b-Amylase enzyme breaks the glucoseeglucose bonds by removing two
glucose units at a time, thereby producing maltose. Amyloglucosidase is the enzyme that
breaks successive bonds from the nonreducing end of the straight chain, producing
glucose. Many microbial amylases usually contain a mixture of these amylases. This
chapter focuses only on a-amylases.
a-Amylases (EC 3.2.1.1) are starch-degrading enzymes that catalyze the hydrolysis of
internal a-1,4-O-glycosidic bonds in the polysaccharides with the retention of the
a-anomeric configuration in the products. Most of the a-amylases are metalloenzymes,
which require calcium ions (Ca2þ) for their activity, structural integrity, and stability.
They belong to family 13 (GH-13) of the glycoside hydrolase group of enzymes [6,7].
Based on the end-product formation a-amylases are classified as saccharifying and
liquefying amylases. The saccharifying a-amylases are further classified as maltose
forming, maltotetraose forming, maltopentaose forming and maltohexaose forming
based on the end products formed [1].
The a-amylase family is the largest family of glycoside hydrolases, transferases, and
isomerases, comprising 30 different enzyme specificities. These enzymes are classified
into four groups: endoamylases, exoamylases, debranching enzymes, and transferases.
Endoamylases are enzymes that cleave internal a-1,4 bonds resulting in a-anomeric
Chapter 1 a-Amylases 5
products. Exoamylases are enzymes that cleave a-1,4, or a-1,6 bonds of the external
glucose residues resulting in a- or b-anomeric products. Debranching enzymes are
enzymes that hydrolyze a-1,6 bonds leaving linear polysaccharides. Transferases are
enzymes that cleave a-1,4 glycosidic bonds of the donor molecule and transfer part of
the donor molecule to a glycosidic acceptor, forming a new glycosidic bond [7].
1.2 Sources of a-Amylase

a-Amylases are universally distributed throughout the plant, animal, and microbial
kingdoms. The enzymes from microbial sources have dominated applications in in-
dustrial processes [2]. Though a-amylases have been derived from several microbial
sources, including bacteria, fungi, yeast, and actinomycetes, the enzymes produced from
bacterial and fungal sources have dominated applications in industrial sectors. Because
of their short growth period, their biochemical diversity, and the ease with which
enzyme concentrations might be increased by environmental and genetic manipulation,
the enzymes from microbial sources generally meet industrial demands.
1.2.1 Plant a-Amylases

Plants store carbon predominantly as starch and the metabolism of starch is essential to
all life. Family 1 a-amylases are characterized by having a secretary signal peptide.
This plays an important role in the degradation of extracellular starch in cereal grain
endosperms. Family 2 a-amylases are characterized by having no predicted targeted
peptide and are localized in the cytoplasm. These amylases have been identified from
monocotyledons, dicotyledons, and gymnosperms. They become most active when the
plastidial starch reserves of leaves are more depleted. They are involved in general stress
responses. Family 3 a-amylases are characterized by having a large N-terminal domain,
which contains a large predicted chloroplast transit peptide. These enzymes are
responsible for degrading plastid-bound starch in storage tissues and leaves [8].
1.2.2 Bacterial a-Amylases

a-Amylases are produced from various bacterial sources, including Bacillus, Brevi-
bacterium, Clostridium, Halomonas, Naxibacter, Nesterenkonia, Paenibacillus,
Pseudomonas, Streptomyces sp., etc. Among the bacterial sources, Bacillus sp. is widely
used, especially for the production of thermostable a-amylases. Bacillus subtilis, Bacillus
stearothermophilus, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus acid-
ocaldarius, Bifidobacterium bifidum, and Bifidobacterium acerans are important sources
used for a-amylase production [9]. Alkaline and thermotolerant amylases have been
reported from Bacillus sp., B. licheniformis, and Bacillus halodurans [10]. Other bacteria
producing a-amylase include Anoxybacillus beppuensis [11], Bacillus laterosporus [12],
Bacillus acidicola [13], Chryseobacterium taeanense [14], Clostridium sp. [15],
Microbacterium foliorum [16], Nesterenkonia sp. [17], Thermococcus sp. [18], Anoxybacillus
flavithermus [19] etc.
1.2.3 Fungal a-Amylases

Several fungal species also produce a-amylases, including Acremonium, Aspergillus,
Penicillium, Mucor, Neurospora, and Thermomyces sp. Among the fungal sources, the
genus Aspergillus has been widely used for the production of a-amylases. Aspergillus
niger, Aspergillus flavus, and Aspergillus oryzae are important sources used among the
fungal sources [20,21]. Other fungal strains producing a-amylase include Thermomyces
lanuginosus [22].
1.3 Production of a-Amylase

1.3.1 Production Methods
To meet the industrial demand, it is essential to develop a low-cost medium for the
production of a-amylase. It can be produced by submerged fermentation (SmF) and
solid-state fermentation (SSF). The production is affected by a variety of physiological
factors, which include pH, temperature, aeration, inoculum concentration, inoculum
age, composition of the growth medium, surfactants, carbon source, nitrogen source,
etc. [23]. Interactions of these parameters have a significant influence on the production
of the enzyme. Generally, SmF is carried out using synthetic media, incorporating me-
dium constituents such as nutrient broth and soluble starch, as well as other compo-
nents, which are very expensive. Replacement of such constituents by cheaper carbon
and nitrogen sources as well as nutrients would benefit the process in cost reduction.
Agricultural by-products offer potential benefits in this regard [7].
SSF is defined as the process in which the growth of microorganisms is carried out on
solid substrates with negligible free water, or free-flowing water [24]. SSF plays an
important role in the production of enzymes. Agro-industrial substrates are considered
the best substrates for SSF processes. It is of special interest in those processes in which a
crude fermented product may be used directly as an enzyme source. The common
substrates used for SSF processes are wheat bran, rice bran, cassava waste, palm oil
waste, banana waste, tea waste, coconut oil cake, coir pith, corn cobs, etc. In SSF, it is
important to provide optimized water content and to control the water activity of the
fermenting substrate. At times, SSF is preferred to SmF because of its simple technique,
low capital investment, lower levels of catabolite repression and end-product inhibition,
low wastewater output, better product recovery, and high-quality production [25].
Continuous and fed-batch studies are more effective for the production of a-amylase.
The study conducted by Lee and Parulekar [26] revealed that the a-amylase production
by B. subtilis TN 106 was enhanced when batch cultivation was extended with fed-batch
cultivation, and the enzyme activity was 54% higher in a two-stage fed-batch operation
compared to a single-stage batch culture. Mishra and Maheswari [27] reported
a-amylase from a thermophilic fungus, T. lanuginosus; the enzyme was a dimeric protein
with a molecular mass of 42 kDa with optimum pH and temperature of 5.6 and 65 C,
respectively. The enzyme produced high levels of maltose from potato starch, suggesting
its usefulness in the commercial production of maltose and glucose syrups. The study
conducted by Krishna and Chandrasekharan [28] revealed that banana peel could be
utilized as a potential substrate for a-amylase production by A. niger. Saxena and Singh
[29] screened various agro-industrial residues for amylase production from Bacillus sp.
and found mustard oil cake to be the best substrate. The strain produced 5400 U/g of
amylase at 1:3 moisture content, 20% inoculum, and an incubation period of 72 h. Yang
and Wang [30] reported a-amylase production by Streptomyces rimosus TM 55 using
sweet potato residue and peanut meal residue as a substrate. The strain produced
1903 U of a-amylase after 96 h of incubation.
Ramachandran et al. [20] used coconut oil cake (COC), a by-product of oil extraction
from dried copra, as a substrate for the production of a-amylase from fungi. COC sup-
plemented with 0.5% starch and 1% peptone enhanced a-amylase production by
A. oryzae. COC serves as a source of soluble proteins and lipids thus providing essential
nutrients for the growth of and enzyme synthesis by the organism. Production of
a-amylase by B. amyloliquefaciens under SSF using corn gluten meal (CGM) was re-
ported by Saban et al. [31]. The study revealed that a-amylase production in a medium
with CGM was five times higher than that in a medium containing starch and other
components. Utilization of CGM as a substrate makes the process economically viable
because CGM is a by-product of starch-based industries.
Production and optimization of a-amylase from A. oryzae CBS 819 using a by-product
of wheat grinding (gruel) as the sole carbon source was done by Kammoun et al. [32].
Various process parameters affecting the production were optimized by adopting a
BoxeBehnken design, which increased the enzyme production from 40.1 to 151.1 U/mL.
Murthy et al. [33] reported coffee by-products as suitable substrate for the production of
a-amylase under SSF. Coffee waste was converted into value-added products by
fermentation using Neurospora crassa CFR 308. The optimum conditions for a-amylase
production were moisture content of 60%, pH 4.5, incubation temperature of 27 C,
particle size of 1 mm, and incubation time of 5 days. Under optimized conditions the
strain produced 7084 U/gds of a-amylase.
Syed et al. [34] reported extracellular amylase production by Streptomyces gulbar-
gensis DAS 131 by SmF. The highest amylase production was observed when the medium
was supplemented with 1% starch. The enzyme was thermotolerant and stable at pH 9.0.
Starch and peptone were good sources of carbon and nitrogen. Sharma and
Satyanarayana [13] reported enhanced production of acidic high-maltose-forming and
Ca2þ-independent a-amylase by B. acidicola; a maximum enzyme titer of 366 IU/L was
attained after 36 h of fermentation at pH 4.5, 33 C, with 0.5 vvm aeration. The enzyme
titer was 10,100 IU/L in fed-batch fermentation. One of the main advantages of fed-
batch fermentation over the batch fermentation is that the concentration of limiting
substrate is maintained at low levels, thus avoiding the repressing effect of high substrate
concentration and thereby minimizing the accumulation of inhibitory metabolites.
A highly thermostable and calcium-independent a-amylase from A. beppuensis

TSSC-1 was reported by Kikani and Singh [11]. This organism produced a monomeric
a-amylase with optimal pH and temperature of 7.0 and 55 C, respectively. The key
findings of this study were cost-effective purification, high thermostability, and broad
pH stability. The enzyme exhibited Ca2þ independence and resistance to chemical
denaturation, which could make it suitable for many industrial applications. Another
agro-industrial residue, date waste, has also been used as the substrate for the pro-
duction of a-amylase using yeast, Candida guilliermondii CGL-A10 [35]. Maximum
enzyme production was attained in SmF (2056 mmol/L/min). Rajagopalan et al. [15]
used sugarcane bagasse hydrolyzate for the production of a-amylase produced by a
solventogenic Clostridium sp. BOH3. The strain used starch directly without any pre-
treatment and produced extracellular amylase (7.15 U/mg protein) and butanol almost
equivalent to 90% of the yield equivalent to glucose. Sugarcane bagasse was used by
Roohi and Kuddus [16] to produce a cold-active a-amylase from M. foliorum GA2.
Maximum enzyme production (6610 U) was observed when fermentation was carried
out in a medium containing 40% bagasse, 0.0003 M lactose, at pH 8.0, with incubation
temperature of 20 C for 5 day at static conditions. This was the first report on cold-
active a-amylase production from M. foliorum GA2. Table 1.1 shows various microor-
ganisms used for the production of a-amylase.
1.3.2 Factors Influencing the Production of a-Amylase

Production of a-amylase by SSF and SmF is affected by a variety of physicochemical
factors [3]. These include media composition, incubation temperature, inoculum age,
carbon source, nitrogen source, pH, phosphate concentration, aeration, and others.
Table 1.1 Strains and Strategies Adopted for a-Amylase Production

Method of
Microorganism Production Substrate Enzyme Yield References
Bacillus subtilis TN 106 Fed batch [26]
Streptomyces rimosus TM55 SSF Sweet potato residue/ 2642.7 U/gds [30]
peanut residue
Aspergillus oryzae SSF Oil cake 9196 U/gds [20]
Aspergillus oryzae OBS819 SmF 151.1 U/mL [32]
Neurospora crassa CFR 308 SSF Coffee waste 7084 U/gds [33]
Streptomyces gulbargensis DAS 131 SmF Starch [34]
Bacillus acidicola SmF 366 IU/L [13]
Anoxybacillus beppuensis TSSC-1 [11]
Candida guilliermondii CGL-A10 SmF 2056 mmol/L/min [35]
Clostridium sp. BOH3 SmF Sugarcane bagasse 7.15 U/mg protein [15]
hydrolyzate
Microbacterium foliorum GA2 SmF Bagasse 6610 U/mL [16]
SmF, submerged fermentation; SSF, solid-state fermentation.

1.3.2.1 Incubation Temperature

The effect of temperature on a-amylase production is related to the growth of the or-
ganism. Temperature control is very important in fermentation processes because
growth and production of enzymes are sensitive to temperature. Hence, the optimum
temperature varies with the culture. a-Amylases have been produced by various mi-
crobes over a wide range of temperature. Productions in SSF as well as in SmF are
usually carried out in the range 25e37 C. However, psychrophilic and thermophilic
temperatures have also been reported for the production. For example, a-amylase
production was attained at 55 C by the thermophilic fungi Thermomonospora fusca [36]
and T. lanuginosus [27] and at 80 C by a hyperthermophilic bacterium, Thermococcus
profundus [36]. A psychrophilic bacterium, Alteromonas haloplanktis, produced
a-amylase at 4 C [37].
1.3.2.2 pH
The pH of the fermentation medium plays an important role in enzyme production. It
induces morphological changes in the organisms as they are sensitive to the concen-
tration of hydrogen ions present in the medium. A pH change in the medium affects the
growth as well as the product stability. Unlike SmF, in which pH control is almost
mandatory for a-amylase production, in SSF processes, generally there is no need to set,
or control, the pH, as the substrates (agro-industrial residues) mostly possess excellent
buffering capacity and keep the pH favorable for the growth and activity of the culture.
Most of the Bacillus strains used commercially for the production of a-amylases have an
optimum pH of 6.0 or 7.0. Some of the medium components eliminate the need for pH
control. Yabuki et al. [38] reported that A. oryzae 557 accumulated a-amylase in the
mycelia when grown in phosphate, or sulfate-deficient, medium and it was released
when the mycelia were placed in a medium with pH above 7.2. Based on the optimal pH
for activity, a-amylases are classified as acidic, neutral, and alkaline [1].
1.3.2.3 Carbon Sources

a-Amylase production could be either constitutive or inducible. Galactose, inulin, and
glycogen are suitable substrates for a-amylase production in SmF. Supplementation with
lactose; an analog of maltose, a-methyl-D-glucoside; and yeast extract induces the
production [7]. Several agro-residues such as wheat bran, rice bran, vegetable peels, fruit
peels, cassava bagasse, and vegetable-oil-extracted residues are used as substrates for
a-amylase production in SSF. Most studies on a-amylase production by A. oryzae suggest
that the general inducer molecule is maltose. Eratt et al. [39] observed a 20-fold increase
in enzyme activity when maltose and starch were used as inducers in A. oryzae NRC
401013. Xylose and fructose support good growth, but they are strongly repressive [40].
1.3.2.4 Nitrogen Sources

Organic as well inorganic nitrogen sources are used for the production of a-amylases,
although organic sources have been preferred over inorganic nitrogen sources. Commonly
used nitrogen sources include bactopeptone, ammonium sulfate, ammonium nitrate, Vogel
salts, casein, meat extract, beef extract, yeast extract, corn steep liquor, and soybean flour.
There are reports on the use of several other nitrogenous sources for a-amylase production.
For example, L-asparagine was reported as the better nitrogen source for enzyme produc-
tion by T. lanuginosus; casein hydrolyzate and yeast extract improved a-amylase
production several fold and by 110e156%, respectively, by A. oryzae [41]. Complex nitrogen
sources in the medium influence the production of a-amylases. Studies carried out by
Dettori et al. [42] revealed that the supplementation of two organic nitrogen sources
enhanced amylase production and this was better than a single organic nitrogen source.
1.3.2.5 Metal Ions

Supplementation of metal ions in the fermentation medium promotes microbial growth,
which in turn accelerates the enzyme production. Most a-amylases are known to be
metal dependent for divalent ions, e.g., Ca2þ, Mg2þ, Mn2þ, and Zn2þ [2].
Supplementation with Ca2þ is generally required for an increased in a-amylase pro-
duction by several bacteria. Ca2þ imparts thermostability of the enzyme due to salting
out of hydrophobic residues by Ca2þ in the protein. The production was reduced to 50%
when Mg2þ was omitted from the medium; Naþ and Mg2þ showed coordinated stimu-
lation of enzyme production by Bacillus sp. CRP strain [43]. However, some metal ions
could have a negative impact on the microbes for a-amylase production, e.g., Li2þ and
Hg2þ have negative effects on a-amylase production. Mg2þ also plays an important role
in a-amylase production.
1.3.2.6 Surfactants
Addition of surfactants to the fermentation medium is generally known to increase the
secretion of proteins by increasing cell membrane permeability. The commonly used
surfactants are Tween 80, Tween 40, Triton X-100, sodium dodecyl sulfate (SDS), poly-
ethylene glycol, and glycerol. These surfactants are reported to increase cell perme-
ability, thereby enhancing enzyme yield. Arneson et al. [44] reported a twofold increase
in a-amylase production by T. lanuginosus. Goes and Sheppard [45] reported a signifi-
cant advantage in using the bio-surfactant surfactin to enhance the production of
a-amylase by B. subtilis in SSF. In addition to increasing the enzyme activity, surfactin
offers other advantages, including eco-friendliness, less sensitivity to extremes of tem-
perature and pH, and being a potential fungicide, thereby eliminating contamination of
the exposed substrate, compared to synthetic surfactants.
1.3.2.7 Agitation
Agitation influences the mixing as well as the oxygen transfer rate in most fermentations
and thus influences cell morphology and product formation [46,47]. It is generally
believed that higher agitation is detrimental to cell growth, which in turn could decrease
enzyme production. Agitation intensities up to 300 rpm are normally employed for the
production of a-amylase in SmF from various microorganisms.
1.4 Assay of a-Amylases

Activity of a-amylases is quantified by measuring either the end products, like glucose or
maltose, or the amount of substrate that remains after enzymatic hydrolysis. a-Amylases
are assayed using soluble starch or modified starch as the substrate. They catalyze the
hydrolysis of a-1,4 glycosidic linkages in starch to produce glucose, dextrins, and limit
dextrins. The reaction is monitored by an increase in the reducing sugar levels or a
decrease in the iodine color of the treated substrate. Various methods are available for the
determination of a-amylase activity [48]. These are based on a decrease in starcheiodine
color intensity, increase in reducing sugars, degradation of color-complexed substrate,
and decrease in viscosity of the starch suspension [3]. The common methods employed
for the determination of a-amylase activity are the iodine method [49]; dextrinizing ac-
tivity [50]; Sandstedt, Kneen, and Blish method [51]; dinitrosalicylic acid method [52];
and degradation of color-complexed substrate [53,54].
The dinitrosalicylic acid (DNS) method [52] is among the most commonly used
methods for estimating the reducing sugars. The DNS reacts with reducing sugar under
boiling and turns to red from yellow. In the method of Fuwa et al. [50], the starch reacts
with iodine and forms a blue solution and the intensity of the color is directly propor-
tional to the starch concentration. Boron dipyrromethene-labeled substrate releases a
fluorescent fragment upon digestion with the enzyme and has been developed for
determining a-amylase activity in foods [55].
1.5 a-Amylase Inhibitors

Proteinaceous a-amylase inhibitors have been isolated from plants and microorganisms
[56]. These inhibitors control endogenous a-amylase activity or work in defense against
pests and pathogens; some inhibitors are antinutritional factors. a-Amylase inhibitors
belong to seven different protein structural families. Six types are from higher plants and
one is from Streptomyces sp. High-resolution structures are available for target
a-amylase and these structures indicate major diversity and some similarities in the
structural basis of a-amylase inhibition. Various types of inhibitors include Streptomyces
inhibitors, knottins, g-thionins, CM proteins, and kunitz-type, thaumatin-like, and
lectin-like inhibitors. Some a-amylase inhibitors have adverse effects on nutrition due to
their inhibition of digestive enzymes in humans and animals. a-Amylase inhibitors find
application in obesity and diabetic therapy.
1.6 Strain Improvement

Strain improvement is usually carried out to increase production as well as to improve
the properties of the enzyme. The catalytic properties of enzymes are determined by
their 3-D structure. Hence, enzyme properties can be altered by site-directed muta-
genesis. Using this method, the properties of an enzyme can be improved, by making
Table 1.2 Some Strategies Adopted for Strain Improvement/Properties of a-Amylase

Microorganism Improved Property References
Bacillus subtilis BR151 Thermostability [60]
Alternaria tenuissima FCBP 252 2.39-fold increased production [62]
Thermobifida fusca NTU22 Increased production [63]
Bacillus amyloliquefaciens Increased production (1.4-fold) [71]
Anoxybacillus sp. High stability in absence of Ca2þ ions at 60 C [66]
and high levels of maltose production
Aspergillus oryzae IIB 30 Increased production (2.1-fold) [70]
Paenibacillus sp. High rate of maltose production [67]
Bacillus licheniformis MSG Self-inducible, catabolite repression free, and [68]
glucose-activated expression system
B. subtilis ASO1a Increased production (7-fold) and high stability [69]
in absence of Ca2þ
Thermotoga maritima Oxidative stability [72]
B. subtilis Improved protein stability and catalytic efficiency [73]
Bacillus sp. AAH-31 Increased production [74]
it thermostable, reducing its dependence on cofactors, or increasing its activity at low

temperature. Studies on the cloning of the a-amylase gene have been extensively carried
out for hyperproduction [7]. Table 1.2 presents some strategies that have been adopted
for strain improvement of a-amylase.
a-Amylases have been engineered for the improvement of properties such as pH
tolerance, thermotolerance, etc. [57e59]. Barnett et al. [58] found that the introduction
of disulfide bonds in the enzymes and alteration of amino acids prone to oxidation by
an amino acid resistant to oxidizing agents improved the stability of the enzyme.
Suzuki et al. [57] constructed hybrids of homologous strains of the B. licheniformis and
B. amyloliquefaciens with improved thermostability. Ozcan and Ozcan [60] introduced
the thermostable plasmid pC194Amy, harboring a 5.2-kb DNA fragment encoding a
gene of B. stearothermophilus, into B. subtilis BR151 by electroporation. The recom-
binant strains produced more thermostable a-amylase compared to the wild-type
strain. A new strain of B. licheniformis CBBD302, carrying a recombinant plasmid,
pHY-amyL, for B. licheniformis a-amylase (BLA) production, was constructed by Niu
et al. [61]. The combination of target-directed screening and genetic recombination led
to an approximately 26-fold improvement in BLA production and export in
B. licheniformis. Shafique et al. [62] reported the production of an extracellular amylase
from Alternaria tenuissima FCBP 252 in SSF. Chemical mutagenesis using ethyl
methanesulfonate (EMS) produced mutants with a high level of a-amylase activity
(2.39-fold) compared to the parental strain. Genetic characterization of the mutants
using random amplified polymorphic DNA PCR revealed that the expression patterns
of the mutants were isogenic variants of the parent strain. Yang et al. [63] expressed
an a-amylase gene from Thermobifida fusca NTU22 in Pichia pastoris X33 because of
its potential application as a food supplement. Recombinant expression resulted in
higher levels of extracellular enzyme production (510 U/L), indicating constitutive
expression and secretion of the protein. The amount of extracellular protein in the
culture of P. pastoris transformants was less than that in the cell-free extract of
Escherichia coli transformants, hence facilitating the application of crude amylase in
industry without purification.
The gene encoding the a-amylase enzyme in B. subtilis PY22 was amplified by PCR,
sequenced, and cloned into P. pastoris KM71H strain using the vector Ppicz A, allowing
methanol-induced expression and secretion of the protein [64]. Recombinant expres-
sion resulted in high levels of extracellular amylase production (22 mg/L). The presence
of Ca2þ ions in the medium resulted in a 41% increase in a-amylase activity. Expression
in P. pastoris not only increased the yield of production but also potentially helped
facilitate purification. Gene cloning and heterologous expression of the high-maltose-
producing a-amylase of Rhizopus oryzae showed successful expression of R. oryzae
a-amylase in P. pastoris at a high level (382 mg/L) [65]. The enzyme had an extremely
high affinity for maltotriose and no maltotriose remained after hydrolysis. Chai et al.
[66] cloned two genes that encoded a-amylases from Anoxybacillus sp. and expressed
them in E. coli. The enzymes produced by the recombinant strains were highly stable
even in the absence of calcium at 60 C for 48 h and they produced high levels of
maltose. Protein sequencing revealed that the recombinant a-amylase differed in 17
amino acids compared to the amylase produced by the wild-type strain. A gene
encoding a-amylase from the genomic DNA of Paenibacillus sp. and the heterologous
expression of recombinant Amy1 in E. coli BL21 (DE3) facilitated the recovery of this
protein in soluble form. The high rate of maltose production due to the action of Amy1
could be exploited for the production of simple sugars as a by-product in food waste
processing [67].
The use of an expression system to overcome catabolite repression opens up an
avenue for exploiting cheap carbon sources for the production of recombinant enzyme.
Nathan and Nair [68] developed a repression-free catabolite-enhanced expression sys-
tem for a thermophilic a-amylase from B. licheniformis MSG. A self-inducible, catabolite
repression-free, and glucose-activated expression system was developed using a ther-
mophilic a-amylase as a model. The a-amylase gene from B. licheniformis MSG without
any 50 cre operator produced unimpeded glucose-enhanced expression when fused to
the phosphate starvation-inducible strong pst promoter with optimum translation sig-
nals in a protease-deficient B. subtilis. The yield was 18.5-fold higher than that of native
promoter. Roy et al. [69] cloned and overexpressed a raw-starch-digesting a-amylase
gene (AmyBS-I) from B. subtilis strain ASO1a in E. coli BL21. The gene also included its
signal peptide sequence for the efficient extracellular expression of recombinant
a-amylase in correctly folded form. The extracellular secretion of AmyBS-I was sevenfold
higher and it did not require Ca2þ ions for its a-amylase activity/thermostability, which
was an added advantage for its use in the starch industry.
Random mutagenesis has also been used for enhanced production of a-amylase. A
strain of A. oryzae IIB 30 was subjected to physical (using UV light) and chemical
mutagenesis (using nitrous acid and EMS). Mutation using EMS-20 showed a 2.1-fold
increased amylase activity compared to the wild-type strain [70]. An identical observa-
tion was earlier reported for a B. amyloliquefaciens strain in which mutation using EMS
improved enzyme activity by 1.4-fold higher than that of the parental strain [71]. Ozturk
et al. [72] reported site-directed mutagenesis of methionine residues for improving the
oxidative stability of a-amylase from Thermotoga maritima. The oxidative stability of
a-amylase (AmyC) was improved by mutating the methionine residues at positions 43
and 44, and 55 and 62, to oxidative-resistant alanine residues. The mutant exhibited
improved oxidative properties. The engineered AmyC could be a potential candidate for
industrial applications, especially in the presence of oxidizing agents. This is the first
protein engineering attempt for AmyC from T. maritima. Yang et al. [73] carried out
structural engineering of histidine residues in the catalytic domain of a-amylase from
B. subtilis for improved protein stability and catalytic efficiency under acidic conditions
by site-directed mutagenesis. The four histidine residues His222, His275, His293, and
His310 in the catalytic domain were selected as the mutation sites and were further
replaced with acidic aspartic acid, respectively yielding four mutants H222D, H275D,
H293D, and H310D. The acidic stability of the enzyme was significantly enhanced after
mutation, and 45e92% of the initial activity of the mutants was retained after incubation
at pH 4.5 and 25 C for 24 h, whereas that for the wild type was only 39.5%. As revealed by
the structure models of the wild-type and mutant enzymes, the hydrogen bonds and salt
bridges were increased after mutation, and an obvious shift of the basic limb toward
acidity was observed for the mutants. These changes around the catalytic domain
contributed to the significantly improved protein stability and catalytic efficiency at low
pH. This work provided an effective strategy to improve the catalytic activity and stability
of a-amylase under acidic conditions, and the results indicated potential application for
the improvement of acid resistance of other enzymes.
The hydrolytic activity of thermophilic, alkalophilic a-amylase could also be
enhanced through the optimization of amino acid residues surrounding the substrate
binding site [74]. Twenty-four selected amino acid residues were replaced with Ala, and
Gly429 and Gly550 were altered to Lys and Glu, respectively, based on comparison of
AmyL’s amino acid sequence with related enzymes. Y426A, H431A, I509A, and K549A
showed higher activity than the wild type at 162e254% of wild-type activity. Tyr426,
His431, and Ile509 were predicted to be located near subsite 2, and Lys549 was near
subsite þ2. Ser, Ala, Ala, and Met were the best amino acid residues for the positions of
Tyr426, His431, Ile509, and Lys549, respectively. Combinations of the optimized single
mutations at distant positions were effective in enhancing catalytic activity. The double-
mutant enzymes Y426S/K549M, H431A/K549M, and I509A/K549M, combining two of
the selected single mutations, showed 340%, 252%, and 271% of wild-type activity,
respectively. Triple- and quadruple-mutant enzymes of the selected mutations did not
show higher activity than the best double mutant, Y426S/K549M.
1.7 Purification and Characterization of a-Amylases

a-Amylases produced by fermentation are relatively crude preparations. Most of the
commercial use of a-amylase does not require 100% purification of the enzyme. But,
high-purity enzymes are required when they are used in clinical and pharmaceutical
sectors. The first steps in the purification involve the isolation of crude enzyme after the
fermentation. In SmF, this is usually done by centrifuging the fermented medium and
taking the supernatant as the source of crude enzyme; in the case of SSF, the fermented
matter is usually mixed with water or buffers, and after suitable mixing the contents are
filtered, whereby the filtrate contains the crude enzyme. Then, the enzyme is concen-
trated (in the supernatant/filtrate), precipitated (using salts/solvents), and purified using
various chromatographic techniques such as ion-exchange chromatography, gel filtra-
tion, isoelectric focusing, etc. Table 1.3 presents strategies adopted for the purification of
a-amylase from various microorganisms.
There are a large number of reports on the purification and characterization of
a-amylases produced by bacterial or fungal sources in SmF and SSF [75e87]. An enzyme
produced in SSF was partially purified by ammonium sulfate fractionation. The enzyme
was optimally active at pH 5.0 and 50 C with a molecular mass of 66 kDa. The presence of
Mn2þ and Fe2þ enhanced the enzyme activity, whereas in the presence of Hg2þ and Cu2þ
the activity was reduced [76]. A partially purified a-amylase from Streptomyces erumpens
MTCC 7317 showed a molecular mass of 54,500 Da [77]. a-Amylase from B. subtilis
KIBGE-HAS was purified by ultrafiltration and ammonium sulfate precipitation with 19.2-
fold purification and specific activity of 4195 U/mg. The enzyme was highly stable in the
presence of various surfactants and detergents. Metal ions such as Mn2þ, Ca2þ, Mg2þ, Kþ,
Co2þ, and Fe3þ activated the enzyme, whereas Ba2þ, Cu2þ, Naþ, and Al3þ strongly
inhibited the activity. A highly efficient raw-starch-digesting a-amylase from B. lichen-
iformis ATCC 9945a was purified by gel-filtration chromatography with a sixfold increase
in specific activity and recovery of 38% with a molecular mass of 31 kDa [82]. The purified
enzyme showed an optimum pH and temperature of 6.5 and 90 C, respectively. Co2þ,
Ni2þ, and Ca2þ slightly stimulated, whereas Hg2þ completely inhibited, a-amylase ac-
tivity. An a-amylase from Brevibacterium linens DSM 20158, purified by ion-exchange
chromatography on a DEAEeSephadex column, showed a 7.88-fold increase in purity
with a 16.80% yield, and it appeared homogeneous on SDSepolyacrylamide gel elec-
trophoresis with a molecular mass of 58 kDa. EDTA and Hg2þ inhibited the enzyme
activity, whereas Mn2þ and Ca2þ enhanced the enzyme activity [83].
A novel SDS- and surfactant-stable, raw-starch-digesting, and halophilic a-amylase
was purified from Nesterenkonia sp. [17]. The extracellular a-amylase was purified to
homogeneity by 80% ethanol precipitation, Q-Sepharose anion-exchange chromatog-
raphy, and Sephacryl S-200 gel-filtration chromatography. The optimum temperature
and pH were 45.8 C and 7.5, respectively. The molecular mass was estimated as 100 kDa.
The enzyme was inhibited by EDTA, but was not inhibited by phenylmethanesulfonyl
fluoride and b-mercaptoethanol. Ca2þ stimulated enzyme activity, whereas the enzyme
Table 1.3 Strategies Adopted for Purification of a-Amylase from Various Microorganisms

Optimum Optimum Molecular
Microorganism Purification Strategy Temperature pH Activators Inhibitors Mass References
Bacillus subtilis ATCC [75]
465
Aspergillus oryzae Ammonium sulfate fractionation 50 C 5.0 Mn2þ and Fe2þ Hg2þ and Cu2þ 66 kDa [76]
Streptomyces erumpens 54.5 kDa [77]
MTCC 7317
B. subtilis KIBGE-HAS Ultrafiltration and ammonium Mn2þ, Ca2þ, Ba2þ, Cu2þ, Naþ, [78]
sulfate precipitation Mg2þ, Kþ, Co2þ, and Al3þ
and Fe3þ
B. subtilis C10 Polyethylene glycol/potassium [79]
phosphate aqueous two-phase
system
Penicillium janthinellum Ammonium sulfate fractionation/ 50 C 5.0 EDTA 42.7 kDa [80]
NCIM 4960 anion-exchange chromatography
(DEAE)
Nesterenkonia sp. 80% ethanol precipitation, Q- 45.8 C 7.5 Ca2þ Fe3þ, Cu2þ, 100 kDa [17]
Sepharose anion-exchange Zn2þ, and Al3þ
chromatography, and Sephacryl S-
200 gel-filtration chromatography
Aspergillus flavus 55 C 5.0 55 kDa [81]
Bacillus licheniformis Gel-filtration chromatography 90 C 6.5 Co2þ, Ni2þ, and Hg2þ 31 kDa [82]
ATCC 9945a Ca2þ
Brevibacterium linens Ion-exchange chromatography on Mn2þ and Ca2þ EDTA and Hg2þ 58 kDa [83]
DSM 20158 a DEAEeSephadex column
Rhizopus microsporus Ammonium sulfate precipitation, 70 C 5.0 75 kDa [84]
Sephadex G25 desalination, and
DEAE-52 cellulose chromatography
Aspergillus oryzae strain Acetone precipitation, size- 50 C 5.6 50 and [85]
S2 exclusion and anion-exchange 42 kDa
chromatography
Penicillium chrysogenum Ammonium sulfate precipitation 60 C 6.0 [86]
and Sephadex G50 filtration
Bacillus 80% ammonium sulfate 70 C 7.0 Mg2þ, Ba2þ, 44 kDa [87]
methylotrophicus strain precipitation, DEAE FF anion Al3þ, and
P11-2 exchange, and Superdex 75 10/ dithiothreitol
300 gel-filtration chromatography
was strongly inhibited by Fe3þ, Cu2þ, Zn2þ, and Al3þ. An aqueous two-phase system
comprising polyethylene glycol/potassium phosphate was used for the partition and
purification of a-amylase from the culture supernatant of B. subtilis C10 [79], which
resulted in a 3.56-fold purification of enzyme with a recovery of 59.37%.
An a-amylase from Penicillium janthinellum NCIM 4960, purified by ammonium
sulfate, showed an almost 20-fold increase in specific activity with a 30.73% yield after
anion-exchange chromatography on DEAE cellulose. The purified enzyme had a mo-
lecular mass of 42.7 kDa. The optimum pH and temperature were 5.0 and 50 C,
respectively. The enzyme showed substrate specificity toward amylose and amylopectin.
The chelating agent EDTA inhibited enzyme activity. The enzyme was stable in the
presence of commercial detergents and stability increased in the presence of CaCl2 [80].
An a-amylase produced by A. flavus isolated from mangrove soil was partially purified
using ammonium sulfate, which resulted in a fivefold increase in enzyme activity. The
partially purified enzyme was optimally active at pH 5.0, temperature of 55 C, with a
molecular mass of 55 kDa. The extracellular amylase was purified by anionic- and
cationic-exchange chromatography and preparative electrophoresis, which resulted in
38-fold purity [81]. Shen et al. [84] purified an acid-stable and thermostable a-amylase
from Rhizopus microsporus isolated from distilled liquor. The crude extract was purified
using ammonium sulfate precipitation, Sephadex G25 desalination, and DEAE-52 cel-
lulose chromatography. The optimum pH and temperature were 5.0 and 70 C, respec-
tively, with a molecular mass of 75 kDa.
1.8 Applications of a-Amylase

a-Amylases find a wide range of biotechnological applications in the textile, food,
pharmaceutical, and detergent industries. With the current developments in biotech-
nology, the applications of a-amylases have been widened to other fields like clinical,
medicinal, and analytical chemistry.
1.8.1 Detergent Applications

a-Amylases comprise one of the ingredients of modern compact detergents. One of the
main advantages of using enzymes in detergents is that much milder conditions can be
used than with enzyme-free detergents [3]. Enzymes help in lowering of the washing
temperature. a-Amylases have been used as powerful laundry detergents since 1975.
Currently, 90% of all commercially available detergents contain a-amylase and the de-
mand for automatic dishwasher detergents is growing. One of the main limitations of
a-amylases in detergents is that it is highly sensitive to calcium and oxidants, which are
components of detergents. However, the development of genetically modified strains for
the production of a-amylases to improve their bleach stability has been achieved by
replacing the oxidation-sensitive amino acids with other amino acids. Replacement
of methionine at position 197 by leucine in B. amyloliquefaciens amylase has resulted in
improved resistance against oxidative compounds [88e91].
1.8.2 Textile Desizing

a-Amylases find application in the textile industry for textile desizing. To protect yarn
from breaking, a removable protective layer is applied to the threads. Starch is an ideal
desizing agent because it is cheap, is readily available, and can be removed very easily
[3]. An effective desizing of starch-sized textiles is achieved by the application of
a-amylases, which selectively remove the sizing and do not attack the fibers. It randomly
cleaves the starch into dextrins, which are water soluble and can be removed by washing.
Amylase from Bacillus sp. has been widely used in textile industries for a long time.
Sarvanan et al. [92] studied the desizing of cotton fabrics using a-amylase from
B. licheniformis. The study revealed that pH and amylase concentration exhibited the
dominant effect, followed by treatment time, on desizing efficiency.
1.8.3 Medicinal and Clinical Chemistry

There are several processes in the medicinal and clinical areas that require the appli-
cation of a-amylases [3]. The first enzyme produced industrially was a fungal amylase in
1894 and was used as a pharmaceutical aid for the treatment of digestive disorders.
Dumoulin et al. [93] observed that the addition of a-amylase to cross-linked amylose
tablets could modulate the release kinetics of the drug.
1.8.4 Paper Industry

a-Amylases find application in the paper and pulp industry for the modification of
starches for coating paper. The coating treatment serves to make the surface of the paper
smooth and strong to improve the writing quality of the paper. Starch acts as a good
sizing agent for the finishing of paper, improving the quality and erasability, in addition
to being a good coating for the paper. The sizing enhances the stiffness and strength of
the paper [94]. Cold-active a-amylase is used in the paper industry because it reduces the
viscosity of starch for the appropriate coating of paper [95].
1.8.5 Starch Liquefaction and Saccharification

One of the most important applications of a-amylase is in starch liquefaction for the
production of glucose and fructose syrups. Starch is converted to high-fructose corn
syrup and is widely used in the beverage industry as a sweetener for soft drinks
because of its high sweetening property. The process requires the usage of highly
thermostable a-amylase for starch liquefaction [3]. The enzymatic conversion involves
three processes, which include gelatinization, liquefaction, and saccharification.
Gelatinization involves dissolution of starch granules, forming a viscous solution;
liquefaction involves partial hydrolysis and leads to a loss in viscosity, followed by
saccharification involving the production of maltose and fructose [96]. The enzymes
from B. licheniformis and B. stearothermophilus are widely used because of their
remarkable thermostability.
1.8.6 Bread and Baking Industry

a-Amylases have been widely used in the baking industry, especially in bread and rolls to
give these products a higher volume, better color, and softer crumb. The enzymes
degrade starch into smaller dextrins, which are subsequently fermented by the yeasts.
This generates additional sugar in the dough, which improves the crust color, taste, and
toasting properties of bread. a-Amylase acts as an antistaling agent and improves the
softness retention and shelf life of baked foods. Generally, a-amylases from
B. stearothermophilus are used commercially in the baking industry [97].
1.8.7 Alcohol Production

For the production of ethanol from starch, it has to be solubilized and then submitted to
two enzymatic steps to obtain fermentable sugars. The conversion process is the
liquefaction, which is carried out by a-amylases, followed by the saccharification using
glucoamylases, leading to the formation of a hydrolyzate containing sugars, which are
then fermented by the yeast to ethanol [98].
1.9 Conclusion and Perspectives

a-Amylases are important enzymes for many industrial processes. Though a-amylases
are produced by several microbes, bacterial amylases are commonly preferred because
of their thermotolerance for several applications. Most of the industrial processes that
involve the usage of a-amylase are carried out under extreme conditions of temperature
and pH. The major challenges in the commercial production of amylases are the yield,
stability, and cost of production. Although considerable successes have been achieved in
the production of a-amylases with increased productivity and desired properties for
industrial applications, to sustain the economic feasibility and newer technological
applications, continued research and technological developments are needed through
biotechnological interventions. To meet the industrial demand, it is important to un-
derstand the structureefunction relationship. With the development and application of
modern techniques such as protein engineering, metabolic engineering, etc., it could be
possible to develop tailor-made a-amylases for application in various sectors. Hence,
research and technological development efforts must to be directed toward the devel-
opment and construction of a-amylases with novel and improved properties.
References
[1] Sharma A, Satyanarayana T. Microbial acid-stable a-amylases: characteristics, genetic engineering
and applications. Process Biochemistry 2013;48:201e11.
[2] Pandey A, Nigam P, Soccol VT, Singh D, Mohan R. Advances in microbial amylases. Biotechnology
and Applied Biochemistry 2000;31:135e52.
[3] Gupta R, Gigras P, Mohapatra H, Goswami VK, Chauhan B. Microbial a-amylases: a biotechno-
logical perspective. Process Biochemistry 2003;38:1599e616.
[4] Lonsane BK, Ramesh MV. Production of bacterial thermostable a-amylase by solid state
fermentation: a potential tool for achieving economy in enzyme production and starch hydro-
lysis. In: Advances in applied microbiology, vol. 35. San Diego: California Academic Press; 1990.
p. 1e56.
[5] Pradeep NV, Ankitha AK, Pooja J. Categorizing phenomenal features of a-amylase (Bacillus
species) using bioinformatic tools. Journal of Advances in Life Science and Technology 2012;4:
27e35.
[6] Bordbar AK, Omidiyan K, Hosseinzadeh R. Study on interactionof a-amylase from Bacillus sub-
tilis with cetyl trimethylammonium bromide. Colloids and Surfaces B: Biointerfaces 2005;40:
67e71.
[7] Sivaramakrishnan S, Gangadharan D, Nampoothiri KM, Soccol CR, Pandey A. a- amylases from
microbial sources e an overview on recent developments. Food Technology and Biotechnology
2006;44:173e84.
[8] Stanley D, Farnden KJF, MacRae EA. Plant a-amylases: functions and roles in carbohydrate
metabolism. Biologia, Bratislava 2005;60:65e71.
[9] Naidu MA, Saranraj P. Bacterial amylase: a review. International Journal of Pharmaceutical and
Biological Archives 2013;4:274e87.
[10] Setyorini E, Takenaka S, Murakami S, Aoki K. Purification and characterization of two novel hal-
otolerant extracellular proteases from Bacillus subtilis strain. Bioscience, Biotechnology and
Biochemistry 2006;70:433e40.
[11] Kikani BA, Singh SP. The stability and thermodynamic parameters of a very thermostable
andcalcium-independent a-amylase from a newly isolated bacterium, Anoxybacillus beppuensis
TSSC-1. Process Biochemistry 2012;47:1791e8.
[12] Manojkumar N, Karthikeyan S, Jayaraman G. Thermostable alpha-amylase enzyme production
from Bacillus laterosporus: statistical optimization, purification and characterization. Biocatalysis
and Agricultural Biotechnology 2013;2:38e44.
[13] Sharma A, Satyanarayana T. Optimization of medium components and cultural variables for
enhanced production of acidic high maltose-forming and Ca2þ independent a-amylase by Bacillus
acidicola. Journal of Bioscience and Bioengineering 2011;111:550e3.
[14] Wang S, Liang Y, Liang T. Purification and characterization of a novel alkali-stable a-amylase from
Chryseobacterium taeanense TKU001, and application in antioxidant and prebiotic. Process
Biochemistry 2011;46:745e50.
[15] Rajagopalan G, He J, Yang KL. Production, purification, and characterization of a-amylase from
solventogenic Clostridium sp. BOH3. Bioenergy Research 2014;7:132e41.
[16] Roohi, Kuddus M. Bio-statistical approach for optimization of cold-active a-amylase production by
novel psychrotolerant M. foliorum GA2 in solid state fermentation. Biocatalysis and Agricultural
Biotechnology 2014;3:175e81.
[17] Shafiei FM, Ziaee A, Amoozegar MA. Purification and biochemical characterization of a novel SDS
and surfactant stable, raw starch digesting, and halophilic a-amylase from a moderately halophilic
bacterium, Nesterenkonia sp. strain. Process Biochemistry 2010;45:694e9.
[18] Jeon E, Jung J, Seo D, Jung D, Holden JF, Park C. Bioinformatic and biochemical analysis of a novel
maltose-forming -amylase of the GH57 family in the hyperthermophilic archaeon Thermococcus sp.
CL1. Enzyme and Microbial Technology 2014;60:9e15.
lu Fincan S, Enez B, Özdemir S, Bekler M. Purification and characterization of thermostable
[19] Agülog
a-amylase from thermophilic Anoxybacillus flavithermus. Carbohydrate Polymers 2014;102:144e50.
[20] Ramachandran S, Patel AK, Nampoothiri KM, Chandran S, Szakacs G, Soccol CR, et al. Alpha
amylase from a fungal culture grown on oil Cakes and its properties. Brazilian Archives of Biology
and Technology 2004;47:309e17.
[21] Sivaramakrishnan S, Gangadharan D, Nampoothiri KM, Soccol CR, Pandey A. Alpha amylase pro-
duction by Aspergillus oryzae employing solid state fermentation. Journal of Scientific and
Industrial Research 2007;66:621e6.
[22] Kunamneni A, Permaul K, Singh S. Amylase production in solid state fermentation by the thermo-
philic fungus Thermomyces lanuginosus. Journal of Bioscience and Bioengineering 2005;100:168e71.
[23] Fogarty WM, Kelly CT. Starch degrading enzymes of microbial origin. Progress in Industrial
Microbiology 1979;15:87e150.
[24] Pandey A, Soccol CR, Leo JAR, Nigam P. Solid state fermentation in biotechnology. New Delhi:
Asiatech Publishers Inc.; 2001. p. 221.
[25] Lonsane BK, Ghildyal NP, Budiatman S, Ramakrishna SV. Engineering aspects of solid-state
fermentation. Enzyme and Microbial Technology 1985;7:258e65.
[26] Lee J, Parulekar SJ. Enhanced production of a-amylase in fed-batch cultures of Bacillus subtilis TN
106 [pAT5]. Biotechnology and Bioengineering 1993;42:1142e50.
[27] Mishra RS, Maheshwari R. Amylases of the thermophilic fungus Thermomyces lanuginosus: their
purification, properties, action on starch and response to heat. Journal of Bioscience 1996;21:653e72.
[28] Krishna C, Chandrasekharan M. Banana waste as a substrate for a- amylase production by Bacillus
subtilis (CBTK 106) under solid state fermentation. Applied Microbiology and Biotechnology 1996;
46:106e11.
[29] Saxena R, Singh R. Amylase production by solid state fermentation of agro-industrial wastes using
Bacillus sp. Brazilian Journal of Microbiology 2011;42:1334e42.
[30] Yang S, Wang J. Protease and amylase production of Streptomyces rimosus in submerged and solid
state cultivation. Botanical Bulletin Academy of Singapore 1999;40:259e65.
[31] Saban TM, Dursan O, Murat E. Production of bacterial a-amylases by Bacillus amyloliquefaciens
under solid substrate fermentation. Biochemical Engineering Journal 2007;37:294e7.
[32] Kammoun R, Naili B, Bejar S. Application of a statistical design to the optimization of parameters
and culture medium for a-amylase production by Aspergillus oryzae CBS 819.72 grown on gruel
(wheat grinding by-product). Bioresource Technology 2008;99:5602e9.
[33] Murthy PS, Naidu MM, Srinivas P. Production of a-amylase under solid-state fermentation utilizing
coffee waste. Journal of Chemical Technology and Biotechnology 2009;84:1246e9.
[34] Syed DG, Agasar D, Pandey A. Production and partial purification of a-amylase from a novel isolate
Streptomyces gulbargensis. Journal of Industrial Microbiology and Biotechnology 2009;36:189e94.
[35] Acourene S, Amourache L, Benchabane A, Djaafri K. Utilization of date wastes as substrate for the
production of a-amylase. International Food Research Journal 2013;20:1367e72.
[36] Chung YC, Kobayasi T, Kannai H, Akiba T, Kudo T. Purification and properties of extracellular
amylase from the hyperthermophilic archeon Thermococcus profundus DT5432. Applied and
Environmental Microbiology 1995;61:1502e6.
[37] Busch JE, Stutzenberger FJ. Amylolytic activity of Thermomonospora fusca. World Journal of
Microbiology and Biotechnology 1997;13:637e42.
[38] Yabuki M, Ono N, Hoshino K, Fukui S. Rapid induction of a-amylase by non-growing mycelia of
Aspergillus oryzae. Applied and Environmental Microbiology 1977;34:1e6.
[39] Eratt JA, Douglas PE, Moranelli F, Seligy VL. The induction of a-amylase by starch in Aspergillus
oryzae: evidence for controlled mRNA expression. Canadian Journal of Biochemistry and Cell
Biology 1984;62:678e90.
[40] Arst HN, Bailey CR. The regulation of carbon metabolism in Aspergillus nidulans. In: Smith JE,
Pateman JA, editors. Genetics and physiology of Aspergillus. New York: Academic Press; 1977.
p. 131e46.
[41] Pederson H, Nielson J. The influence of nitrogen sources on the a-amylase productivity of
Aspergillus oryzae in continuous cultures. Applied Microbiology and Biotechnology 2000;53:
278e81.
[42] Dettori BG, Priest FG, Stark JR. Hydrolysis of starch granules by the amylase from Bacillus stear-
othermophilus NCA 26. Process Biochemistry 1992;27:17e21.
[43] Wu WX, Mabinadji J, Betrand TF, Wu WX. Effect of culture conditions on the production of an
extracellular thermostable alpha-amylase from an isolate of Bacillus sp. Journal of Zhejiang
University of Agricultural Life Science 1999;25:404e8.
[44] Arnesen S, Eriksen SH, Olsen J. Increased production of amylase from Thermomyces lanuginosus by
the addition of Tween 80. Enzyme and Microbial Technology 1998;23:249e52.
[45] Goes AP, Sheppard JD. Effect of surfactants on a-amylase production in a solid substrate fermen-
tation process. Journal of Chemical Technology and Biotechnology 1999;74:709e12.
[46] Bhavaraju SM, Blanch HW. A model for pellet breakup in fungal fermentations. Journal of
Fermentation Technology 1976;54:466e8.
[47] Amanullah A, Blair R, Nienow AW, Thomas CR. Effects ofagitation intensity on mycelial morphology
and protein production in chemostat cultures of recombinant Aspergillus oryzae. Biotechnology
and Bioengineering 1999;62:434e46.
[48] Priest FG. Extracellular enzyme synthesis in the genus Bacillus. Bacteriological Reviews 1977;41:
711e53.
[49] Hollo J, Szeitli J. The reaction of starch with iodine. In: Rodley JA, editor. Starch and its derivatives.
4th ed. Chapman & Hall; 1968. p. 203e46.
[50] Fuwa H. A new method for microdetermination of amylase activity by the use of amylose as sub-
strate. Journal of Biochemistry 1954;41:583e603.
[51] Sandstedt RM, Kneen E, Blish MJ. A standardised Wohlgemuth procedure for a-amylase activity.
Cereal Chemistry 1939;16:712e23.
[52] Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical
Chemistry 1959;31:426e8.
[53] Ceska M, Hultman E, Ingelman BGA. A new method for determination of a-amylase. Experentia
1969;25:555e6.
[54] Dhawale MR, Wilson JJ, Khachatourians GG, Ingledew WM. Improved method for detection of
starch hydrolysis. Applied and Environmental Microbiology 1982;44:747e50.
[55] Koyama K, Hirao T, Toriba A, Hayakawa K. An analytical method for measuring a- amylase activity
in food starch-containing foods. Biomedical Chromatography 2013;5:583e8.
[56] Svensson B, Fukuda K, Nielsen PK, Bønsager BC. Proteinaceous a-amylase inhibitors. Biochimica et
Biophysica Acta 2004;1696:145e56.
[57] Suzuki Y, lto N, Yuuki T, Yamagata H, Udaka S. Amino acid residues stabilizing a Bacillus aeamylase
against irreversible thermoinactivation. Journal of Biological Chemistry 1989;203:18933e8.
[58] Barnett CC, Mitchinson C, Power SD, Roquadt CA. Oxidatively stable alpha-amylases. Patent
application US. 5:824e532. 1998.
[59] Nielsen JE, Borchert TV. Protein engineering of bacterial a-amylases. Biochimica et Biophysica Acta
2000;1543:253e74.
[60] Ozcan BD, Ozcan BD. Expression of thermostable a-amylase gene from Bacillus stearothermophilus
in various Bacillus subtilis strains. Annals of Microbiology 2008;58:265e7.
[61] Niu D, Zuo Z, Shi1 G, Wang Z. High yield recombinant thermostable a-amylase production using an
improved Bacillus licheniformis system. Microbial Cell Factories 2009;8:1e7.
[62] Shafique S, Bajwa R, Shafique S. Mutation of Alternaria tenuissima FCBP-252 for hyperactive
a-amylase. Indian Journal of Experimental Biology 2009;47:591e6.
[63] Yang C, Huang Y, Chen C, Wen C. Expression of Thermobifida fusca thermostable raw starch
digesting alpha-amylase in Pichia pastoris and its application in raw sago starch hydrolysis. Journal
of Industrial Microbiology and Biotechnology 2010;37:401e6.
_
[64] Karakas B, Inan M, Certel M. Expression and characterization of Bacillus subtilis PY22 a-amylase in
Pichia pastoris. Journal of Molecular Catalysis B: Enzymatic 2010;64:129e34.
[65] Li S, Zuo Z, Niu D, Singh S, Permaul K, Prior BA, et al. Gene cloning, heterologous expression and
characterization of a high maltose-producing a-amylase of Rhizopus oryzae. Applied Biochemistry
and Biotechnology 2011;164:581e92.
[66] Chai YY, Rahman RN, Illias RM, Goh KM. Cloning and characterization of two new thermostable
and alkalitolerant a-amylases from the Anoxybacillus species that produce high levels of maltose.
Journal of Industrial Microbiology 2012;39:731e41.
[67] Rajesh T, Kim YH, Choi Y, Jeon JM, Kim HJ, Park S, et al. Identification and functional character-
ization of an a-amylase with broad temperature and pH stability from Paenibacillus sp. Applied
Biochemistry and Biotechnology 2013;170:359e69.
[68] Nathan S, Nai M. Engineering a repression-free catabolite-enhanced expression system for
a thermophilic alpha-amylase from Bacillus licheniformis. Journal of Biotechnology 2013;168:394e402.
[69] Roy JK, Borah A, Mahant CL, Mukherjee CK. Cloning and overexpression of raw starch digesting
a-amylase gene from Bacillus subtilis strain AS01a in Escherichia coli and application of the purified
recombinant a-amylase (AmyBS-I) in raw starch digestion and baking industry. Journal of
Molecular Catalysis B: Enzymatic 2013;97:118e29.
[70] Abdullah R, Haq I, Iftikhar T, Butt ZA, Khattak MI. Random mutagenesis for enhanced production
of alpha amylase by Aspergillus oryzae IIB-30. Pakistan Journal of Botany 2013;45:269e74.
[71] Haq I, Javed AMM, Hameed U, Saleem A, Adnan F, Qadeer MA. Production of alpha amylase from a
randomly induced mutant strain of Bacillus amyloliquefaciens and its application as a desizer in
textile industry. Pakistan Journal of Botany 2010;42:473e84.
[72] Ozturk H, Ece S, Gundeger E, Evran S. Site-directed mutagenesis of methionine residues for
improving the oxidative stability of a-amylase from Thermotoga maritima. Journal of Bioscience
and Bioengineering 2013;116:449e51.
[73] Yang H, Liu L, Shin H, Chen RR, Li J, Du G, Chen J. Structure-based engineering of histidine residues
in the catalytic domain of a-amylase from Bacillus subtilis for improved protein stability and cat-
alytic efficiency under acidic conditions. Journal of Biotechnology 2013;164:59e66.
[74] Tamamuraa N, Saburia W, Mukaia A, Morimotob N, Takehanab T, Koikeb S, et al. Enhancement of
hydrolytic activity of thermophilic alkalophilic a-amylase from Bacillus sp. AAH-31 through opti-
mization of aminoacid residues surrounding the substrate binding site. Biochemical Engineering
Journal 2014;86:8e15.
[75] Nabatchian F, Farzami B. Purification of a-amylase from Bacillus subtilis ATCC 465. Journal of the
Islamic Republic of Iran 1993;6:281e4.
[76] Patel AK, Nampoothiri KM, Ramachandran S. Partial purification and characterization of a-amylase
produced by Aspergillus oryae using spent-brewing grains. Indian Journal of Biotechnology 2005;4:
336e41.
[77] Kar S, Ray RC. Partial characterization and optimization of extracellular thermostable Ca2þ
inhibited a-amylase production by Streptomyces erumpens MTCC 7317. Journal of Scientific and
Industrial Research 2008;67:58e64.
[78] Bano S, Quader SAU, Aman A, Azhar A. Partial purification and some properties of a-amylase from
Bacillus subtilis KIBGE-HAS. Indian Journal of Biochemistry and Biophysics 2009;46:401e4.
[79] Loc NH, Mien NTT, Thuy DTB. Purification of extracellular a-amylase from Bacillus subtilis by
partitioning in polyethylene glycol/potassium phosphate aqueous two phase system. Annals of
[80] Sindhu R, Suprabha GN, Shashidhar S. Purification and characterization of a-amylase from
Penicillium janthinellum (NCIM 4960) and its application in detergent industry. Biotechnology
Bioinformatics and Bioengineering 2010;1:37e45.
[81] Bhattacharya S, Bhardwaj S, Das A, Anand S. Utilization of sugarcane bagasse for solid- state
fermentation and characterization of a- amylase from Aspergillus flavus isolated from Muthupettai
mangrove, Tamil Nadu, India. Australian Journal of Basic and Applied Sciences 2011;5:1012e22.
[82] Bozic N, Ruiz J, Santin J, Vujcic Z. Production and properties of the highly efficient raw starch digesting
a-amylase from a Bacillus licheniformis ATCC 9945. Biochemical Engineering Journal 2011;53:203e9.
[83] Shabbiri K, Adnan A, Noor B, Jamil S. Optimized production, purification and characterization of
alpha amylase by Brevibacterium linens DSM 20158, using bio-statistical approach. Annals of
[84] Shen H, Mo X, Chen X, Han D, Zhao C. Purification and enzymatic identification of an acid stable
and thermostable a-amylase from Rhizopus microsporus. Journal of the Institute of Brewing 2012;
118:309e14.
[85] Sahnoun M, Bejar S, Sayari A, Triki MA, Kriaa M, Kammoun R. Production, purification and
characterization of two a-amylase isoforms from a newly isolated Aspergillus oryzae strain S2.
Process Biochemistry 2012;47:18e25.
[86] Doss A, Anand SP. Purification and optimization of fungal amylase from litter samples of Western
Ghats, Coimbatore, Tamilnadu (India). Journal of Scientific Research and Reviews 2013;2:001e4.
[87] Xie F, Quan S, Liu D, Ma H, Li F, Zhou F, et al. Purification and characterization of a novel a-amylase
from a newly isolated Bacillus methylotrophicus strain P11-2. Process Biochemistry 2014;49:47e53.
[88] Svendsen A, Bisgaard-Frantzen H. PCT patent publication. WO 94/0 1994.
[89] Tierny L, Danko S, Dauberman J, Vaha-Vahe P, Winetzky D. Performance advantages of novel
a-amylases in automatic dishwashing. In: Am oil Chem Soc 86th san Antonio Annual meeting; 1995.
[90] Bisgaard-Frantzen H, Borchert T, Svendsen A, Thellersen MH, Van Der Zee P. PCT Patent
Application. WO 95/10603 1995.
[91] Kiran KK, Chandra TS. Production of surfactant and detergent-stable, halophilic, and alkali tolerant
alpha-amylase by a moderately halophilic Bacillus sp. Strain TSCVKK. Applied Microbiology and
Biotechnology 2008;77:1023e31.
[92] Saravana D, Prakash AA, Jagadeeshwaran D. Optimization of thermophilic Bacillus licheniformis
a-amylase desizing of cotton fabrics. Indian Journal of Fiber and Textile Research 2011;36:253e8.
[93] Dumoulina Y, Cartiliera L, Mateescub M. Cross-linked amylose tablets containing a-amylase: an
enzymatically-controlled drug release system. Journal of Controlled Release 1999;60:161e7.
[94] Bruinenberg PM, Hulst AC, Faber A, Voogd RH. A process for surface sizing or coating of paper.
1996. European Patent Application, EP 0 690 170 A1.
[95] Kuddus M. Microbial cold-active a-amylases: from fundamentals to recent developments. Current
Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology
2010:1265e76.
[96] de Souza PM, de Oliveira Magalhães P. Application of microbial a-amylase in industry e a review.
Brazilian Journal of Microbiology 2010;41:850e61.
[97] van der Maarel MJ, van der Veen B, Uitdehaag JC, Leemhuis H, Dijkhuizen L. Properties and ap-
plications of starch-converting enzymes of the alpha-amylase family. Journal of Biotechnology
2002;94:137e55.
[98] Öner ET. Optimization of ethanol production from starch by an amylolytic nuclear petite
Saccharomyces cerevisiae strain. Yeast 2006;23:849e56.
2
Amylolytic Enzymes: Glucoamylases
S. Negi*, K. Vibha
MOTILAL NEHRU NATIONAL INSTITUTE OF TECHNOLOGY , A LLAHABAD , IND IA
2.1 Introduction
Starch is one of the most abundant polymers on earth and its industry has a big stake in
the market. Starch is composed of unbranched amylose and branched amylopectin,
which require three types of amylolytic enzymes for complete hydrolysis into glucose,
i.e., a-amylase (4-a-D-glucan glucanohydrolase, EC 3.2.1.1), b-amylase (4-a-D-glucan
maltohydrolase, EC 3.2.1.2), and glucoamylase (4-a-D-glucan glucohydrolase, EC 3.2.1.3).
a-Amylase cleaves the a-1,4-D-glucosidic linkages between adjacent glucose units in the
linear amylose chain; b-amylase cleaves at nonreducing chain ends of amylose,
amylopectin, and glycogen molecules; and GA hydrolyzes a-1,4 glycosidic bonds from
the nonreducing ends of starch and a-1,6 linkages at the branching points of amylo-
pectin, although at a lower rate than 1,4 linkages, into glucose [1e4].
GA can also catalyze the reverse hydrolysis reaction to produce maltose and iso-
maltose, which has great significance in industrial processes in which high sugar content
is present. GA converts starch and a- and b-limit dextrins into glucose and shows faster
reaction on polysaccharides than on oligosaccharides. The rate of hydrolysis depends on
the substrate size and the structure, nature, and position of the bond present. GA is
ubiquitously present in or produced by all forms of life (plants, animals, bacteria, archaea,
and eukaryotes). However it is mainly produced using filamentous fungi, although a host
of other microorganisms are also known as good producers of GA. Aspergillus niger,
Aspergillus awamori, and Rhizopus oryzae are the commonly used filamentous fungi for
industrial production of GA [9,10]. GAs are extensively used in the food and beverage
industries. They are used for production of glucose syrup, high-fructose corn syrup, beer,
soy sauce, alcoholic beverages, etc. [2,9,11e13].
Most of the GA produced from parent strains catalyzes saccharification efficiently only
within a small range of mild temperatures. At high temperatures its catalytic
activity reduces sharply because of conformation changes. GA produced from parent
fungal sources normally has limited thermostability, catalytic activity, and low pH range,
which restrict its application in industrial processes carried out at high temperature and
in alkaline medium. At higher temperature the reaction rate is higher; therefore,
*
Corresponding Author.
Current Developments in Biotechnology and Bioengineering: Production, Isolation and Purification of Industrial Products
http://dx.doi.org/10.1016/B978-0-444-63662-1.00002-6 25
processing is faster. It also prevents microbial contamination and reduces the viscosity of
the reaction mixture. This leads to reduction in process cost. Production of GA that is
stable at higher temperatures would be highly beneficial for starch saccharification.
Advances in recombinant DNA technology and site-directed and random mutagenesis
and other techniques are being used to improve the thermostability and other functional
properties of GA [1]. Over the years a lot of research has been carried out to reduce the
cost of production of GA and improve its functional properties to suit industrial re-
quirements. Progress in the fields of molecular biology, protein engineering, and bioin-
formatics has helped to provide it with improved functional properties, such as enhanced
thermostability, better selectivity, wider pH range, improved catalytic activity, etc. [14].
2.2 Sources of Glucoamylase

GA occurs in a wide range of organisms. It is present in plants [15], animals [16], fungi,
bacteria, and yeast. However, microorganisms are the main source explored for GA
production.
2.2.1 Microbial Sources

GA is present in a wide range of bacteria, fungi, and yeasts. Some of the microbial
sources exploited for the production of GA are shown in Table 2.1.
2.2.1.1 Fungal Sources

Fungal species of Aspergillus, Rhizopus, and Endomyces are the most commonly used
sources for production of GA. Aspergillus awamori and A. niger are among the most
popular microorganisms used by industry for GA production [2,3,17]. Rhizopus oryzae
[18], Rhizopus niveus [19], Mucor [6,20], Penicillium [21], and many other fungal species
are capable of producing GA. Enzyme production by molds is generally extracellular,
which makes its downstream processing cost-effective and less time consuming.
2.2.1.2 Bacterial Sources

There are many bacterial strains capable of producing GA. Flavobacterium sp. [22],
Bacillus stearothermophilus [23], Sclerotinia sclerotiorum [24], Sclerotium rolfsii [25], and
Thermoanaerobacter tengcongensis [26] are some of the bacteria used for production of
GA. The amylolytic bacterial strains Clostridium thermosaccharolyticum [27], Clostridium
sp. [28], and Lactobacillus amylovorus [29] have also been used for production of GA [2].
Thermostable GA from thermophilic bacteria can be used at higher temperature for
saccharification, thereby reducing the production cost of glucose by saving the cost of
cooling. For production of glucose from starch, liquefaction of starch is done by
a-amylase first, followed by saccharification by GA. Liquefaction can take place rapidly
at 95e105 C by a thermostable bacterial a-amylase. But fungal GAs are stable normally
up to a temperature range of 55e60 C. Therefore, most of the fungal GAs are used only
Chapter 2 Amylolytic Enzymes: Glucoamylases 27
Table 2.1 Details of Substrates and Microorganisms Used for Production of

Glucoamylase
Source Microorganism Process Substrate Yield References
Fungal Aspergillus oryzae SSF Corn flour, 5582.4 mmol/m g [37]
FK-923 wheat bran
Fungal A. oryzae SSF Wheat bran 45.21 U/mL [38]
Fungal Aspergillus niger SSF Wheat bran 2.0 and 1.99 mmol/mL min [5]
and Rhizopus
Fungal A. oryzae SSF Wheat bran and rice 4.1 IU [39]
bran
Fungal A. niger SSF Potato starch 31.214 U/mg [7]
Fungal A. niger SSF Potato starch 152.85 U/mL [40]
Fungal A. oryzae SSF Wheat bran and 330 mg/mL min [41]
sugarcane bagasse
Fungal Aspergillus niveus SmF Starch and yeast 600 U/mg of protein [42]
extract
Fungal Aspergillus awamori SSF Potato starch 8.3 U/mL [9]
Fungal Thermomucor SSF Sucroseeyeast extract 26.3 U/mL [43]
indicae-seudaticae
Fungal A. awamori SSF Wheat bran 9420.6 U/gds [44]
Fungal Aspergillus flavus SmF Cassava and corn [45]
and Thermomyces starch
lanuginosus
Fungal A. niger SSF Rice bran 695 U/g [46]
Fungal T. lanuginosus SSF Starch 60 U/mg of protein [47]
Fungal Colletotrichum SSF Starch 0.45 IU/mL [4]
gloeosporioides
Bacterial Thermoanaerobacter SSF Maltose 80 U/mg [26]
tengcongensis
Bacterial Bacillus sp. SSF Starch 21.6 U/mL [30]
Bacterial Lactobacillus amylovorus SSF Dextrin and starch [29]
Yeast Candida famata SSF Starch 2587.17 mmol/L m [31]
Yeast Pichia subpelliculosa SmF Commercial washed 6500 U/L [32]
starch
Plant Sugar beet Liquid MurashigeeSkoog 102.2 U/mg [36]
(Beta vulgaris L.) medium
SmF, submerged fermentation; SSF, solid-state fermentation.
after the reaction mixture is cooled down to this temperature. Thermostable GA from
thermophilic bacteria can be used for saccharification without much cooling of the
liquefied starch. GA produced from aerobic bacteria, such as B. stearothermophilus,
Halobacterium sodomense, and Flavobacterium sp., and anaerobic bacteria, such as
Clostridium sp. and C. thermosaccharolyticum, have better thermostability compared to
fungal GA [26].
A thermostable GA (TtcGA) from T. tengcongensis was successfully expressed in

Escherichia coli by Zheng et al. [26]. Heat treatment and gel-filtration chromatography
were used to partially purify the recombinant mature protein, and 30-fold homogeneity
was obtained. Maximum activity of the recombinant enzyme was obtained at 75 C and
pH 5.0. It was highly thermostable with almost no activity loss at 75 C for 6 h. James and
Lee (1995) used L. amylovorus to produce GA in dextrose-free de ManeRogosaeSharpe
medium in a 1.5-L fermenter under an optimal dextrin concentration of 1% (w/v), pH
5.5, and 37 C. GA production was maximum at the late logarithmic phase of growth for
16e18 h. Crude enzyme showed maximum activity at pH 6.0 and 60 C. Gill and Kaur
(2004) used a bacterial strain of Bacillus for production of TtcGA [30]. Highest produc-
tion of GA was achieved at 65 C and pH 7.0 after 17e20 h under stationary conditions.
Luria broth, supplemented with 0.5% (w/v) soluble starch, yielded highest enzyme ac-
tivity (21.6 U/mL). GA showed optimum activity at 70 C and pH 5.0. It was highly stable
at pH 7.0 with a half-life of 13 h, 8 h, and 3 h 40 min at 60, 65, and 70 C, respectively.
2.2.1.3 Yeast Sources

Candida famata [31], Pichia subpelliculosa [32], and Saccharomyces diastaticus [33] are
some of the yeast used for production of GA. GA production has also been reported from
yeast strains of Saccharomycopsis fibuligera [34] and Lipomyces starkeyi HN-606 [35].
Mohamed et al. isolated C. famata from traditional Moroccan sourdough for production
of GA [31]. Starch enhanced GA production, with maximum GA activity at 5 g/L. Yeast
extract and (NH4)2HPO4 gave maximum GA and biomass after 72 h of incubation in
liquid medium at 30 C, pH 5, at 105 rpm.
2.2.2 Other Sources

There are very few reports of GA production from plant and animal origins. In one such
work on GA production from a plant origin, sugar beet (Beta vulgaris L.) was explored for
production of GA and a-amylase [36]. They reported the presence of GA and a-amylase
in callus and suspension cultures as well as in mature roots of sugar beets (B. vulgaris L.).
2.3 Glucoamylase Production

GA is produced by many microorganisms capable of growing in a vast variety of sub-
strates through the fermentation process. Like any other enzyme production, GA also
depends on the selection of microbial strain, substrate, medium, and fermentation
process and on physicochemical parameters such as incubation time, temperature, pH,
relative humidity [in solid-state fermentation (SSF)], inhibitors, oxygen accessibility, etc.
Production can be carried out through submerged fermentation (SmF) or SSF depending
upon the microbial culture and substrate in use. Until recently, approximately 90% of
all industrial enzymes were produced in SmF using a specifically optimized process
and genetically manipulated microorganisms. However, scientists have discovered and
Chapter 2 Amylolytic Enzymes: Glucoamylases 29
realized the numerous economical and practical advantages of SSF. Almost all enzymes
can be produced in SSF using microorganisms. Some of the sources and GA production
details are shown in Table 2.1.
2.3.1 Selection of Fermentation Process

In SmF the substrate is solubilized or suspended as fine particles in a large volume of
liquid, whereas in SSF an insoluble substrate is fermented with sufficient moisture but
with no free water. SSF is an ideal process when the organism is a filamentous fungus,
which is able to withstand the limited water availability. SSF has become popular with
scientists as well as industry, particularly with agro-based substrates [2,48,49]. SSF has
distinct advantages over SmF in terms of lower production costs, lower energy re-
quirements, higher yield, simple fermentation equipment, and less effluent generation.
In SSF, nutrients present in the substrate serve as an anchorage for the microbial cells. In
SSF the moisture content varies from 30% to 80% and maximum enzyme production is
normally obtained with about 60% moisture content. At lower moisture content
microbiological activity ceases.
2.3.2 Composition of Substrate

Composition of the substrate used for production of GA has a big impact on its yield.
Selection of substrate is done considering its availability and suitability for the particular
strain and process used. Agro-industrial residues like rice husk, wheat bran, rice bran,
gram flour, coconut oil cake, sugarcane bagasse, wheat flour, corn flour, tea waste,
potato starch, etc., are commonly used as substrates for GA production in SSF
[2,17,38,49,50]. Corn starch is a commonly used substrate for production of glucose
syrup in the United States and Europe because of its easy availability. The particle size of
the substrate has a strong influence on the growth rate and enzyme-producing ability of
the organism due to the influence of surface area on growth rates. The optimum particle
size is to be decided to ensure maximum mass transfer because bigger particles provide
less surface area and smaller particles provide a high degree of solubilization. Pandey
reported poor GA activity with a particle size of larger than 1.4 mm and smaller than
180 mm [50]. Wheat bran particles of 425e500 mm were found most suitable for maxi-
mizing GA activity. Another vital factor affecting GA production in SSF is water activity,
which is an important parameter for mass transfer of the water and solutes across the
cell membrane. Pandey et al. reported higher GA yield with higher initial water activity
values of substrate [51].
Substrates are normally supplemented with carbon or nitrogen sources to increase the
enzyme yield. Supplement can be a simple carbon source like glucose, maltose, or su-
crose or a polymeric compound such as starch from some other source, or a nitrogen
source such as ammonium or nitrate salts or urea, or a complex source, e.g., corn steep
liquor. The requirements for nutrition are normally more complex in SmF compared to
SSF. Supplements of fructose, ammonium sulfate, urea, and yeast extract in the substrate,
Another random document with
no related content on Scribd:
be severely injured, and until all hopes of her ever becoming a
mother are at an end.
158. The quiet retirement of her own home ought then to be her
greatest pleasure and her most precious privilege. Home is, or ought
to be, the kingdom of woman, and she should be the reigning
potentate. England is the only place in the world that truly knows
what home really means. The French have actually no word in their
language to express its meaning:
“That home, the sound we English love so well,
Has been as strange to me as to those nations
That have no word, they tell me, to express it.”[33]
159. Cheerfulness, contentment, occupation, and healthy activity

of mind cannot be too strongly recommended. A cheerful, happy
temper is one of the most valuable attributes a wife can have. The
possession of such a virtue not only makes herself, but every one
around her, happy. It gilds with sunshine the humblest dwelling, and
often converts an indifferent husband into a good one. Contentment
is the finest medicine in the world; it not only frequently prevents
disease, but, if disease be present, it assists in curing it. Happy is the
man who has a contented wife! A peevish, discontented helpmate
(helpmate, save the mark!) is always ailing, is never satisfied, and
does not know, and does not deserve to know, what real happiness is.
She is “a thorn in the flesh.”
160. One of the greatest requisites, then, for a happy home is a
cheerful, contented, bright, and merry wife; her face is a perpetual
sunshine, her presence is that of an angel; she is happy in herself,
and she imparts happiness to all around her. A gentle, loving,
confiding, placid, hopeful, and trusting disposition has a great charm
for a husband, and ought, by a young wife, to be assiduously
cultivated—
“For gentleness, and love, and trust
Prevail o’er angry wave and gust.”[34]
161. Every young wife, let her station be ever so exalted, ought to
attend to her household duties. Her health, and consequently her
happiness, demand the exertion. The want of occupation—healthy,
useful occupation—is a fruitful source of discontent, of sin,[35] of
disease, and barrenness. If a young married lady did but know the
importance of occupation—how much misery might be averted, and
how much happiness might, by attending to her household duties, be
insured—she would appreciate the importance of the advice.
Occupation improves the health, drives away ennui, cheers the
hearth and home, and, what is most important, if household duties
be well looked after, her house becomes a paradise, and she the
ministering angel to her husband. But she might say—I cannot
always be occupied; it bores me; it is like a common person: I am a
lady; I was not made to work; I have neither the strength nor the
inclination for it; I feel weak and tired, nervous and spiritless, and
must have rest. I reply, in the expressive words of the poet, that—
“Absence of occupation is not rest,—
A mind quite vacant is a mind distress’d.”[36]
“If time be heavy on your hands,” are there no household duties to

look after, no servants to instruct, no flower-beds to arrange, no
school children to teach, no sick-room to visit, no aged people to
comfort, no widow nor orphan to relieve?—
“Nor any poor about your lands?
Oh! teach the orphan boy to read,
Or teach the orphan girl to sew—
Pray Heaven for a human heart.”[37]
162. To have nothing to do is most wretched, wearisome, and

destructive to the mind. The words of Martin Luther on this subject
should be written in letters of gold, and ought to be kept in constant
remembrance by every man and woman, be they rich or poor,
lettered or unlettered, gentle or simple. “The mind,” said he, “is like a
mill that cannot stop working; give it something to grind, and it will
grind that. If it has nothing to grind, it grinds on yet, but it is itself it
grinds and wears away.”
163. A lady in this enlightened age of ours considers it to be
horribly low and vulgar to strengthen her loins with exercise and her
arms with occupation, although such a plan of procedure is
recommended in the Bible by the wisest of men,—“She girdeth her
loins with strength, and strengthened her arms.”[38]
164. A husband soon becomes tired of grand performances on the
piano, of crotchet and worsted work, and of other fiddle-faddle
employments; but he can always appreciate a comfortable, clean,
well-ordered, bright, cheerful, happy home, and a good dinner. It
might be said that a wife is not the proper person to cook her
husband’s dinner. True; but a wife should see and know that the cook
does her duty; and if she did, perchance, understand how the dinner
ought to be cooked, I have yet to learn that the husband would for
such knowledge think any the worse of her.
165. A grazing farmer is three or four years in bringing a beast to
perfection, fit for human food. Is it not a sin, after so much time and
pains, for an idiot of a cook, in the course of one short hour or two, to
ruin, by vile cookery, a joint of such meat? Is it not time, then, that a
wife herself should know how a joint of meat ought to be cooked, and
thus to be able to give instructions accordingly?
166. A boy is brought up to his profession, and is expected to know
it thoroughly; how is it that a girl is not brought up to her profession
of a wife; and why is it that she is not taught to thoroughly
understand all household duties? The daughters of a gentleman’s
family in olden time spent an hour or two every morning in the
kitchen and in the laundry, and were initiated into the mysteries of
pastry and pudding-making, of preserving fruit, of ironing, etc. Their
mothers’ and their grandmothers’ receipt-books were at their finger-
ends. But now look at the picture; the daughters of a gentleman’s
family of the present day consider it very low and horridly vulgar to
understand any such matters. It is just as absurd to ask a lady to play
on the piano who has never been taught music as to ask a wife to
direct her servants to perform duties which she herself knows
nothing about. The duties of a wife cannot come either by intuition
or by instinct more than music can. Again I say, every lady, before
she be married, ought to be thoroughly taught her profession—the
duties of a wife; she then would not be at the tender mercies of her
servants, many of whom are either unprincipled or inefficient.
167. Do not think that I am overstating the importance of the
subject. A good dinner—I mean a well-cooked dinner (which, be it
ever so plain, is really a good dinner)—is absolutely essential to the
health, to the very existence of yourself and your husband; and how,
if it be left to the tender mercies of the present race of cooks, can you
have it? High time it is that every wife, let her station be either high
or low, should look into the matter herself, and remedy the crying
evil of the day. They manage these things better in Sweden. There the
young ladies of wealthy families cook—actually themselves cook—the
dinners; and instead of their considering it a disgrace, and to be
horridly low and vulgar, they look upon it as one of their greatest
privileges! And what is the consequence? A badly-cooked dinner is
rare, and not, as it frequently is in this country, of frequent
occurrence; and “peace and happiness” reign triumphant. It is a pity,
too, that we do not take a leaf out of the book of our neighbors the
French. Every woman in France is a good cook; good cookery with
them is the rule—with us it is the exception. A well-cooked dinner is
a blessing to all who partake of it; it promotes digestion, it sweetens
the temper, it cheers the hearth and home. There is nothing tries the
temper more than an ill-cooked dinner; it makes people dyspeptic,
and for a dyspeptic to be sweet-tempered is an utter impossibility.
Let me, therefore, advise my fair reader to look well into the matter;
either the gloom or the sunshine of a house much depends upon
herself and upon her household management. It might be said—
What a poor creature a man must be to require so much attention.
Truly, if his health be not looked after, if his comforts be not
attended to, he is indeed a poor creature!
168. Every young wife should be able—ought to be instructed by
her mother or by some competent person—it should be a part of her
education—to teach and to train her own servants aright.
Unfortunately, in the present day there is too much cant and
humbug about the instruction of the lower orders, and domestic
servants among the rest. They are instructed in many things that are
perfectly useless to them, the knowledge of which only makes them
dissatisfied with their lot and tends to make them bad servants.
Among other useless subjects taught them are the “ologies.” It would
be much more to the purpose if they were thoroughly instructed in
all household duties, and “in the three R’s—reading, ’riting, and
’rithmetic,”—in obedience to their mistresses, and in simplicity of
demeanor and dress. The servants themselves would be immensely
benefited by such lessons.
169. A “blue-stocking” makes, as a rule, a wretched wife; it would
be far better for the health of her husband, of herself, and her family,
if, instead of cultivating Latin and Greek, she would cultivate her
household duties, more especially a thorough knowledge of the
culinary department. “A man is, in general, better pleased when he
has a good dinner upon his table than when his wife speaks
Greek.”[39]
170. As soon as a lady marries, the romantic nonsense of school-
girls will rapidly vanish, and the stern realities of life will take their
place, and she will then know, and sometimes to her grievous cost,
that a useful wife will be thought much more of than either an
ornamental or a learned one.
171. It is better for a young wife, and for every one else, to have too
much than too little occupation. The misfortune of the present day is,
that servants are made to do all the work, while the mistress of the
house remains idle. Remains idle! Yes; and by remaining idle,
remains out of health! Idleness is a curse, and brings misery in its
train! How slow the hours crawl on when a person has nothing to do;
but how rapidly they fly when she is fully occupied! Besides, idleness
is a frequent cause of barrenness. Hard-worked, industrious women
are prolific; while idle ladies are frequently childless, or, if they do
have a family, their children are puny, and their labors are usually
both hard and lingering. We doctors know full well the difference
there often is between the labor of a poor hard-worked woman and of
a rich, idle lady: in the one case the labor is usually quick and easy; in
the other, it is often hard and lingering. Oh, if wives would consider
betimes the importance of an abundance of exercise and of
occupation, what an immense amount of misery, of pain, of anxiety,
and anguish they might avert! Work is a blessed thing; if we do not
work we pay the penalty—we suffer “in mind, body, and estate.” An
idle man or an idle woman is an object of the deepest pity and
commiseration.
172. Longfellow, in his Song of the Blacksmith, beautifully and
graphically describes the importance and the value of occupation;
and as occupation is as necessary to a woman as to a man, I cannot
resist transcribing it:
“Toiling—rejoicing—sorrowing,
Onward through life he goes;
Each morning sees some task begin,
Each evening sees its close;
Something attempted, something done,
Has earned a night’s repose.”
173. Truly may it be said that “occupation earns a night’s repose.”

It is the finest composing medicine in the world, and, unlike an
opiate, it never gives a headache; it never produces costiveness; and
never, by repetition, loses its effect. Sloth and restlessness, even on
down, are generally bed-fellows:
“Weariness
Can snore upon the flint, when rusty sloth
Finds the down pillow hard.”
174. The mind, it is well known, exerts great influence over the
body in promoting health, and in causing and in curing disease. A
delicate woman is always nervous; she is apt to make mountains of
molehills; she is usually too prone to fancy herself worse than she
really is. I should recommend my gentle reader not to fall into this
error, and not to magnify every slight ache or pain. Let her, instead
of whining and repining, use the means which are within the reach of
all to strengthen her frame; let her give battle to the enemy; let her
fight him with the simple weapons indicated in these pages, and the
chances are she will come off victorious.
175. There is nothing like occupation, active occupation, to cure
slight pains—“constant occupation physics pain”—to drive away little
ailments, and the dread of sickness. “The dread of sickness,” says Dr.
Grosvenor, “is a distemper of itself, and the next disposition to a
many more. What a bondage does this keep some people in! ’Tis an
easy transition from the fear and fancy of being sick to sickness
indeed. In many cases there is but little difference between those
two. There is one so afraid of being ill that he would not stir out of
doors, and for want of air and exercise he contracts a distemper that
kills him.”
176. What a blessed thing is work! What a precious privilege for a
girl to have a mother who is both able and anxious to instruct her
daughter, from her girlhood upwards, in all household management
and duties! Unfortunately, in this our age girls are not either
educated or prepared to be made wives—useful, domesticated wives.
Accomplishments they have without number, but of knowledge of
the management of an establishment they are as ignorant as the babe
unborn. Verily, they and their unfortunate husbands and offspring
will in due time pay the penalty of their ignorance and folly! It is,
forsooth, unladylike for a girl to eat much; it is unladylike for her to
work at all; it is unladylike for her to take a long walk; it is unladylike
for her to go into the kitchen; it is unladylike for her to make her own
bed; it is unladylike for her to be useful; it is unladylike for her to
have a bloom upon her cheek like unto a milkmaid![40] All these are
said to be horridly low and vulgar, and to be only fit for the common
people! Away with such folly! The system of the bringing up of the
young ladies of the present day is “rotten to the core.”
177. If a young married lady, without having any actual disease
about her, be delicate and nervous, there is no remedy equal in value
to change of air—more especially to the sea-coast. The sea breezes,
and, if she be not pregnant, sea-bathing, frequently act like magic
upon her in restoring her to perfect health. I say, if she be not
pregnant; if she be, it would, without first obtaining the express
permission of a medical man, be highly improper for her to bathe.
178. A walk on the mountains is delightful to the feelings and
beneficial to the health. In selecting a sea-side resort, it is always,
where it be practicable, to have mountain-air as well as the sea
breeze. The mounting of high hills, if a lady be pregnant, would not
be desirable, as the exertion would be too great, and, if she be
predisposed, might bring on a miscarriage; but the climbing of hills
and mountains, if she be not enceinte, is most advantageous to
health, strengthening the frame, and exhilarating to the spirits.
Indeed, we may compare the exhilaration it produces to the drinking
of champagne, with this difference,—it is much more beneficial to
health than champagne, and does not leave, the next morning, as
champagne sometimes does, either a disagreeable taste in the mouth
or headache behind,—
“Oh, there is a sweetness in the mountain-air,
And life, that bloated ease can never hope to share!”[41]
179. Bugs and fleas.—This is a very commonplace subject, but like

most commonplace subjects is one necessary to be known, as these
pests of society sometimes destroy the peace, comfort, and
enjoyment of a person when away from home. Many ladies who
travel from home are made miserable and wretched by having to
sleep in strange beds—in beds infested either with bugs or with fleas.
Now, it will be well for such ladies never to go any distance from
home without having four things in their trunks with them, namely:
(1) A box of matches, in order, at any moment of the night, to strike a
light, both to discover and frighten the enemies away. (2) A box of
night-lights. Bugs never bite when there is a light in the room. It
would therefore be well, in an infested room, and until fresh lodgings
can be procured, to keep a night-light burning all night. (3) A packet
of “La Poudre Insecticide,” manufactured in France, but which may
be procured in England: a preparation which, although perfectly
harmless to the human economy, is utterly destructive to fleas. (4) A
4 oz. bottle of oil of turpentine, a little of which, in case of a discovery
of bugs in the bed, should be sprinkled between the sheets and on the
pillow. The oil of turpentine will, until fresh lodgings can be
procured, keep the bugs at a respectful distance. Care should be
observed while sprinkling the sheets with the turpentine not to have
(on account of its inflammability) a lighted candle too near the bed. I
know, from experience, that bugs and fleas are, when ladies are away
from home, a source of torment and annoyance, and am therefore
fully persuaded of the value and importance of the above advice.
180. If it be not practicable for her to visit the sea-coast, let her be
in the fresh air—in the country air. Let her mornings be spent out of
doors; and if she cannot inhale the sea breezes, let her inhale the
morning breezes—
“The skies, the air, the morning’s breezy call
Alike are free, and full of health to all.”[42]
181. Cheerfulness and evenness of temper ought, by a young wife,

to be especially cultivated. There is nothing that promotes digestion,
and thus good health, more than a cheerful, placid temper. We know
that the converse is very detrimental to that process; that violent
passion takes away the appetite, deranges the stomach, and
frequently disorders the bowels. Hence it is that those who attain
great ages are usually of an even, cheerful temper. “Our passions are
compared to the winds in the air, which, when gentle and moderate,
let them fill the sail, and they will carry the ship on smoothly to the
desired port; but when violent, unmanageable, and boisterous, it
grows to a storm, and threatens the ruin and destruction of all.”[43]
182. A young wife is apt to take too much opening medicine; the
more she takes, the more she requires. Hence she irritates the nerves
of the stomach and bowels, and injures herself beyond measure. If
the bowels are costive, and variety of food, and of fruit, and of other
articles of diet, which I either have or will recommend in these pages,
together with an abundance of air, and of exercise, and of
occupation, will not open, then let her give herself an enema; which
she can, without the slightest pain or annoyance, and with very little
trouble, readily do, provided she has a proper apparatus for the
purpose, namely, a “self-injecting enema apparatus,”—one made
purposely for the patient, either to administer it to herself, or to be
administered to her by another person. A pint of cold water is as
good an enema as can be used, and which, if the first should not
operate, ought in a few minutes to be repeated. The clyster does
nothing more than wash the bowels out, removing any offending
matter, and any depression of spirits arising therefrom, and neither
interfering with the stomach nor with the digestion.
183. Until she become accustomed to the cold, she might for the
first few mornings slightly warm the water; but gradually she should
reduce the temperature of it until she use it quite cold. A cold water
is more bracing and strengthening to the bowels, and more
efficacious in action, than a warm water enema.
184. It will, during pregnancy and after a confinement, be safer to
use a tepid than a cold water enema.
185. No family ought to be without a good enema apparatus, to fly
to in any emergency. Many valuable lives have been saved by means
of it, and having it always in good order and at hand.
186. By adopting the dictates of reason and of common sense,
many of the nervous, useless, lackadaisical, fine ladies will be
unknown; and we shall have instead blooming wives, who will in due
time become the mothers of hardy, healthy, happy children.
187. In the foregoing pages the burden of my song has been health
—the preservation of health—the most precious of God’s gifts, and
one that is frittered and fooled away as though it were but of little
value. Health ought to be the first consideration of all, and of every
young wife especially, as, when she is married, her life, her health is
not altogether her own, but her husband’s and her family’s. Oh! it is a
glorious gift, a precious boon, to be in the enjoyment of perfect
health, and is worth a little care and striving for.
188. In concluding the first division of my subject, let me entreat
my fair reader to ponder well on what I have already said; let her
remember that she has a glorious mission; let her thoroughly
understand that if good habits and good rules be not formed and
followed during the first year of her wifehood, they are not at all
likely to be instituted afterwards. The first year, then, is the golden
opportunity to sow the seeds of usefulness; to make herself healthy
and strong, and to cause her to be a blessing, a solace, and a comfort
to her husband, her children, and all around her.
189. Menstruation, during a period of about thirty years, plays a
momentous part in the female economy; indeed, unless it be in every
way properly and duly performed, it is neither possible that such a
lady can be well, nor is it at all probable that she will conceive. I
therefore purpose devoting an especial chapter to its due and careful
consideration.
PART I.
MENSTRUATION.
190. There are two most important epochs in the life of a woman—
namely (1) the commencement, and (2) the close of menstruation.
Each is apt, unless carefully watched and prevented, to bring in its
train many serious diseases. Moreover, unless menstruation be
healthfully and properly performed, conception, as a rule, is not
likely to take place: hence the importance of our subject.
191. Menstruation—the appearance of the catamenia or the menses
—is then one of the most important epochs in a girl’s life. It is the
boundary line, the landmark, between childhood and womanhood; it
is the threshold, so to speak, of a woman’s life. Her body now
develops and expands, and her mental capacity enlarges and
improves. She then ceases to be a child, and she becomes a woman.
She is now for the first time, as a rule, able to conceive.
192. Although puberty has at this time commenced, it cannot be
said that she is at her full perfection; it takes eight or ten years more
to complete her organization, which will bring her to the age of
twenty-three or twenty-five years; which perhaps are the best ages
for a woman, if she have both the chance and the inclination, to
marry.
193. If she marry when very young, marriage weakens her system,
and prevents a full development of the body. Besides, if she marry
when she be only eighteen or nineteen, the bones of the pelvis—the
bones of the lower part of the belly—are not at that time sufficiently
developed; are not properly shaped for the purpose of labor; do not
allow of sufficient space for the head of the child to readily pass, as
though she were of the riper age of twenty-three or twenty-five. She
might have in consequence a severe and dangerous confinement. If
she marry late in life, say after she be thirty, the soft parts engaged in
parturition are more rigid and more tense, and thus become less
capable of dilatation, which might cause, for the first time, a hard
and tedious labor. Again, when she marries late in life, she might not
live to see her children grow up to be men and women. Moreover, as
a rule, “the offspring of those that are very young or very old lasts
not.” Everything, therefore, points out that the age above indicated—
namely, somewhere between twenty and thirty—is the most safe and
suitable time for a woman to marry.
194. Menstruation generally comes on once every month—that is
to say, every twenty-eight days; usually to the very day, and
frequently to the hour. Some ladies, instead of being “regular” every
month, are “regular” every three weeks.
195. Each menstruation continues from three to five days; in some
for a week; and in others for a longer period. It is estimated that,
during each menstruation, from four to six ounces is, on an average,
the quantity discharged.
196. A lady seldom conceives unless she be “regular,” although
there are cases on record where women have conceived who have
never been “unwell;” but such cases are extremely rare.
197. Menstruation in this country usually commences at the ages
of from thirteen to sixteen, sometimes earlier; occasionally as early
as eleven or twelve; at other times later, and not until a girl be
seventeen or eighteen years of age. Menstruation in large towns is
supposed to commence at an earlier period than in the country, and
earlier in luxurious than in simple life.[44]
198. Menstruation continues for thirty, and sometimes even for
thirty-five years; and, while it lasts, is a sign that a lady is liable to
become pregnant—unless, indeed, menstruation should be
protracted much beyond the usual period of time. As a rule, then,
when a woman “ceases to be unwell,” she ceases to have a family;
therefore, as menstruation usually leaves her at forty-five, it is
seldom, after that age, that she has a child.
199. I have known ladies become mothers when they have been
upwards of fifty years of age. I myself delivered a woman in her fifty-
first year of a fine healthy child. She had a kind and easy labor, and
was the mother of a large family, the youngest being at the time
twelve years old.[45] “Dr. Carpenter, of Durham, tells us that he has
attended in their confinements several women whose ages were fifty.
‘I well recollect a case occurring in my father’s practice in 1839,
where a woman became a widow at forty-nine years of age. Shortly
afterwards she married her second husband, and within twelve
months of this time gave birth to her first child. These cases belong
to the working classes. But I know of two others, where gentlewomen
became mothers at fifty-one with her first child, the other with her
eighth. I can say nothing of how they menstruated, but I know of a
virgin in whom the catamenia appeared regularly and undiminished
up to and at the end of sixty.’ Dr. Powell says that he last year
attended a woman in her fifty-second year; and Mr. Heckford, that
he attended a woman who stated her age to be at least fifty. Mr.
Clarke, of Mold, states that he has attended several women whose
ages were upwards of forty-four, and that he lately delivered a
woman of her first child at forty-eight. Mr. Bloxham, of Portsmouth,
delivered at fifty-two, in her first confinement, a woman who had
been married thirty-five years.”[46]
200. In very warm climates, such as in Abyssinia and in India, girls
menstruate when very young—at ten or eleven years old; indeed,
they are sometimes mothers at those ages.[47] But when it commences
early, it leaves early; so that they are old women at thirty. “Physically,
we know that there is a very large latitude of difference in the periods
of human maturity, not merely between individual and individual,
but also between nation and nation—differences so great that in
some southern regions of Asia we hear of matrons at the age of
twelve.”[48] Dr. Montgomery[49] brings forward some interesting cases
of early maturity. He says: “Bruce mentions that in Abyssinia he has
frequently seen mothers of eleven years of age; and Dunlop
witnessed the same in Bengal. Dr. Goodeve, Professor of Midwifery
at Calcutta, in reply to a query on the subject, said: ‘The earliest age
at which I have known a Hindu woman bear a child is ten years, but I
have heard of one at nine.’”
201. In cold climates, such as Russia, women begin to menstruate
late in life, frequently not until they are between twenty and thirty
years old; and, as it lasts on them thirty or thirty-five years, it is not
an unusual occurrence for them to bear children at a very advanced
age—even so late as sixty. They are frequently not “regular” oftener
than three or four times a year, and when it does occur the menstrual
discharge is generally sparing in quantity.
202. The menstrual fluid is not exactly blood, although, both in
appearance and in properties, it much resembles it; yet it never in
the healthy state clots as blood does. It is a secretion from the womb,
and, when healthy, ought to be of a bright-red color, in appearance
very much like blood from a recently cut finger.[50]
203. The menstrual fluid ought not, as before observed, to clot. If
it does, a lady, during menstruation, suffers intense pain; moreover,
she seldom conceives until the clotting has ceased. Application must
therefore, in such a case, be made to a medical man, who will soon
relieve the above painful symptoms, and, by doing so, will probably
pave the way to her becoming pregnant.
204. Menstruation ceases entirely in pregnancy, during suckling,
and usually both in diseased and in disordered states of the womb. It
also ceases in cases of extreme debility, and in severe illness,
especially in consumption; indeed, in the latter disease—
consumption—it is one of the most unfavorable of the symptoms.
205. It has been asserted, and by men of great experience, that
sometimes a woman menstruates during pregnancy. In this assertion
I cannot agree; it appears utterly impossible that she should be able
to do so. The moment she conceives, the neck of the womb becomes
plugged up by means of mucus; it is, in fact, hermetically sealed.
There certainly is sometimes a slight red discharge, looking very
much like menstrual fluid, and coming on at her monthly periods;
but being usually very sparing in quantity, and lasting only a day or
so, and sometimes only for an hour or two; but this discharge does
not come from the cavity of, but from some small vessels at, the
mouth of the womb, and is not menstrual fluid at all, but a few drops
of real blood. If this discharge came from the cavity of the womb, it
would probably lead to a miscarriage. My old respected and talented
teacher, the late Dr. D. D. Davis,[51] declared that it would be quite
impossible during pregnancy for menstruation to occur. He
considered that the discharge which was taken for menstruation
arose from the rupture of some small vessels about the mouth of the
womb.
206. Some ladies, though comparatively few, menstruate during
suckling; when they do, it may be considered not the rule, but the
exception. It is said, in such instances, that they are more likely to
conceive. Many persons have an idea that when a woman, during
lactation, menstruates, the milk is both sweeter and purer. Such is an
error. Menstruation during suckling is more likely to weaken the
mother, and consequently to deteriorate the milk. It therefore
behooves a parent never to take a wet nurse who menstruates during
the period of suckling.
207. A lady sometimes suffers severe pains both just before and
during her “poorly” times. When such be the case, she seldom
conceives until the pain be removed. She ought therefore to apply to
a medical man, as relief may soon be obtained. When she is freed
from the pain, she will, in all probability, in due time become
enceinte.
208. If a married woman have painful menstruation, even if she
become pregnant, she is more likely, in the early stage, to miscarry.
This is an important consideration, and requires the attention of a
doctor.
209. If a single lady, who is about to be married, have painful
menstruation, it is incumbent on either her mother or a female
friend to consult, two or three months before the marriage takes
place, an experienced medical man, on her case; if this be not done,
she will most likely, after marriage, either labor under ill health, or
be afflicted with barrenness, or, if she do conceive, be prone to
miscarry.
210. The menstrual discharge, as before remarked, ought, if
healthy, to be of the color of blood—of fresh, unclotted blood. If it be
either too pale (and it sometimes is almost colorless), or, on the
other hand, if it be both dark and thick (it is occasionally as dark, and
sometimes nearly as thick, as treacle), there will be but scant hopes
of a lady conceiving. A medical man ought, therefore, at once to be
consulted, who will in the generality of cases, be able to remedy the
defect. The chances are, that as soon as the defect be remedied, she
will become pregnant.
211. Menstruation at another time is too sparing; this is a frequent
cause of a want of family. Luckily a doctor is, in the majority of cases,
able to remedy the defect, and by doing so will probably be the
means of bringing the womb into a healthy state, and thus
predispose her to become a mother.
212. A married lady is very subject to the “whites;” the more there
will be of the “whites” the less there will usually be of the menstrual
discharge;—so that in a bad case of the “whites” menstruation might
entirely cease, until proper means be used both to restrain the one
and to bring back the other. Indeed, as a rule, if the menstrual
discharge, by proper treatment, be healthily established and
restored, the “whites” will often cease of themselves. Deficient
menstruation is a frequent cause of the “whites,” and the consequent
failure of a family; and as deficient menstruation is usually curable, a
medical man ought, in all such cases, to be consulted.
213. Menstruation at other times is either too profuse or too long
continued. Either the one or the other is a frequent source of
barrenness, and is also weakening to the constitution, and thus tends
to bring a lady into a bad state of health. This, like the former cases,
by judicious management may generally be rectified; and being
rectified, will in all probability result in the wife becoming a mother.
214. When a lady is neither pregnant nor “regular,” she ought
immediately to apply to a doctor, as she may depend upon it there is
something wrong about her, and that she is not likely to become
enceinte[52] until menstruation be properly established. As soon as
menstruation be duly and healthily established, pregnancy will most
likely, in due time, ensue.
215. When a lady is said to be “regular,” it is understood that she is
“regular” as to quality, and quantity, and time. If she be only
“regular” as to the time, and the quantity be either deficient or in
excess, or if she be “regular” as to the time, and the quality be bad,
either too pale or too dark; or if she be “regular” as to the quality and
quantity, and be irregular as to the time, she cannot be well; and the
sooner means are adopted to rectify the evil, the better it will be for
her health and happiness.
216. There is among young wives, of the higher ranks, of the
present time, an immense deal of hysteria; indeed it is, among them,
in one form or another, the most frequent complaint of the day. Can
it be wondered at? Certainly not. The fashionable system of spending
married life, such as late hours, close rooms, excitement, rounds of
visiting, luxurious living, is quite enough to account for its
prevalence. The menstrual functions in a case of this kind are not
duly performed; she is either too much or too little “unwell;”
menstruation occurs either too soon, or too late, or at irregular
periods. I need scarcely say that such a one, until a different order of
things be instituted, and until proper and efficient means be used to
restore healthy menstruation, is not likely to conceive; or, if she did
conceive, she would most likely either miscarry, or, if she did go her
time, bring forth a puny, delicate child. A fashionable wife and happy
mother are incompatibilities! Oh, it is sad to contemplate the
numerous victims that are sacrificed yearly on the shrine of fashion!
The grievous part of the business is, that fashion is not usually
amenable to reason and common sense; argument, entreaty, ridicule,
are each and all alike in turn powerless in the matter. Be that as it
might, I am determined boldly to proclaim the truth, and to make
plain the awful danger of a wife becoming a votary of fashion.
217. Many a lady, either from suppressed or from deficient
menstruation, who is now chlorotic, hysterical, and dyspeptic, weak
and nervous, looking wretchedly, and whose very life is a burden,
may, by applying to a medical man, be restored to health and
strength.
218. As soon as a lady “ceases to be after the manner of women”—
that is to say, as soon as she ceases to menstruate—it is said that she
has “a change of life;” and if she does not take care, she will soon
have “a change of health” to boot, which, in all probability, will be for
the worse.
219. After a period of about thirty years’ continuation of
menstruation, a woman ceases to menstruate; that is to say, when
she is about forty-four or forty-five years of age, and, occasionally, as
late in life as when she is forty-eight years of age, she has “change of
life,” or, as it is sometimes called, a “turn of years.” Now, before this
takes place, she oftentimes becomes very “irregular;” at one time she
is “regular” before her proper period; at another time either before or
after; so that it becomes a dodging time with her, as it is so styled. In
a case of this kind menstruation is sometimes very profuse; at
another it is very sparing; occasionally it is light colored, almost
colorless; sometimes it is as red as from a cut finger; while now and
then it is as black as ink.
220. When “change of life” is about, and during the time, and for
some time afterwards, a lady labors under, at times, great flushings
of heat; she, as it were, blushes all over; she goes very hot and red,
almost scarlet; then perspires; and afterwards becomes cold and
chilly. These flushings occur at very irregular periods; they might
come on once or twice a day, at other times only once or twice a
week, and occasionally only at what would have been her “poorly
times.” These flushings might be looked upon as rather favorable
symptoms, and as an effort of nature to relieve itself through the
skin. These flushings are occasionally, although rarely, attended with
hysterical symptoms. A little appropriate medicine is for these
flushings desirable. A lady while laboring under these heats is
generally both very much annoyed and distressed; but she ought to
comfort herself with the knowledge that they are in all probability
doing her good service, and that they might be warding off, from
some internal organ of her body, serious mischief.
221. “Change of life” is one of the most important periods of a
lady’s existence, and generally determines whether, for the rest of
her days, she shall either be healthy or otherwise; it therefore
imperatively behooves her to pay attention to the subject, and in all
cases when it is about taking place to consult a medical man, who
will, in the majority of cases, be of great benefit to her, as he will be
able to ward off many important and serious diseases to which she
would otherwise be liable. When “change of life” ends favorably,
which, if properly managed, it most likely will do, she may improve
in constitution, and may really enjoy better health and spirits, and
more comfort, then she has done for many previous years. A lady
who has during the whole of her wifehood eschewed fashionable
society, and who has lived simply, plainly, and sensibly, and who has
taken plenty of out-door exercise, will, during the autumn and winter
of life, reap her reward by enjoying what is the greatest earthly
blessing—health!
PART II.
PREGNANCY.
SIGNS OF PREGNANCY.
222. The first sign that leads a lady to suspect that she is pregnant
is her ceasing to be unwell. This, provided she has just before been in
good health, is a strong symptom of pregnancy; but still there must
be others to corroborate it.
223. The next symptom is morning sickness. This is one of the
earliest symptoms of pregnancy; as it sometimes occurs a few days,
and indeed generally not later than a fortnight or three weeks, after
conception. Morning sickness is frequently distressing, oftentimes
amounting to vomiting, and causing a loathing of breakfast. This sign
usually disappears after the first three or four months. Morning
sickness is not always present in pregnancy; but, nevertheless, it is a
frequent accompaniment; and many who have had families place
more reliance on this than on any other symptom.
224. A third symptom is shooting, throbbing, and lancinating
pains, and enlargement of the breasts, with soreness of the nipples,
occurring about the second month; and in some instances, after the
first few months, a small quantity of watery fluid, or a little milk, may
be squeezed out of them. This latter symptom, in a first pregnancy, is
valuable, and can generally be relied on as conclusive that the female
is pregnant. It is not so valuable in an after pregnancy, as a little milk
might, even should she not be pregnant, remain in the breasts for
some months after she has weaned her child.
225. The veins of the breast look more blue, and are consequently
more conspicuous than usual, giving the bosom a mottled
appearance. The breasts themselves are firmer and more knotty to
the touch. The nipples, in the majority of cases, look more healthy
than customary, and are somewhat elevated and enlarged; there is

Current Developments in Biotechnology and Bioengineering. Production, Isolation and Purification of Industrial Products 1st Edition Ashok Pandey

Uploaded by

Copyright:

Available Formats

Current Developments in Biotechnology and Bioengineering. Production, Isolation and Purification of Industrial Products 1st Edition Ashok Pandey

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Current Developments in Biotechnology and Bioengineering. Production, Isolation and Purification of Industrial Products 1st Edition Ashok Pandey

Uploaded by

Copyright:

Available Formats

Download More ebooks [PDF]. Format PDF ebook download PDF KINDLE.

Full download ebooks at ebookmass.com

Current Developments in Biotechnology

Download More ebooks from https://ebookmass.com

Current Developments in Biotechnology and

Current Developments in Biotechnology and

Current Developments in Biotechnology and

Current Developments in Biotechnology and

Current Developments in Biotechnology and

Current Developments in Biotechnology and

Current Developments in Biotechnology and

Current Developments in Biotechnology and

AMSTERDAM l BOSTON l HEIDELBERG l LONDON l NEW YORK l OXFORD

Copyright © 2017 Elsevier B.V. All rights reserved.

British Library Cataloguing-in-Publication Data

For information on all Elsevier publications

Publisher: John Fedor

Typeset by TNQ Books and Journals

M. Adsul DBT-IOC Centre for Advanced Bioenergy Research, IndianOil Corporation

Cristóbal N. Aguilar Food Research Department, School of Chemistry,

A. Angel-Cuapio Universidad Autónoma Metropolitana-Iztapalapa, Mexico City,

G.S. Anisha Government College for Women, Trivandrum, Kerala, India

P. Binod CSIR-National Institute for Interdisciplinary Science and Technology (NIIST),

J. Buenrostro-Figueroa Department of Biotechnology, Division of Health and

S. Chakraborty Indian Institute of Technology Guwahati, Guwahati, Assam, India

M.L. Chávez González Food Research Department, School of Chemistry,

G.-Q. Chen Tsinghua University, Beijing, China

S. Chen Hubei University, Wuhan, PR China

Juan C. Contreras-Esquivel Food Research Department, School of Chemistry,

J.D. Coral Bioprocess Engineering and Biotechnology Department, Federal

J.C. de Carvalho Bioprocess Engineering and Biotechnology Department, Federal

J. de Oliveira Bioprocess Engineering and Biotechnology Department, Federal

A. Dhillon Indian Institute of Technology Guwahati, Guwahati, Assam, India

M.J. Fernandes Bioprocess Engineering and Biotechnology Department, Federal

R. Gaur Indian Oil Corporation Limited, R&D Centre, Faridabad, India

A. Goyal Indian Institute of Technology Guwahati, Guwahati, Assam, India

L.R.C. Guimarães Bioprocess Engineering and Biotechnology Department, Federal

M. Haridas Kannur University, Kannur, India

R. Hemamalini Indian Institute of Technology Delhi, New Delhi, India

Ayerim Hernandez-Almanza Food Research Department, School of Chemistry,

A. Illanes Pontiﬁcia Universidad Católica de Valparaíso, Valparaíso, Chile

J. Isar University of Delhi South Campus, New Delhi, India

A. Joseph Kannur University, Kannur, India

S.G. Karp Bioprocess Engineering and Biotechnology Department, Federal

N. Karthik CSIR-National Institute for Interdisciplinary Science and Technology

R. Kaushik University of Delhi South Campus, New Delhi, India

S.K. Khare Indian Institute of Technology Delhi, New Delhi, India

P.C.S. Kirnev Bioprocess Engineering and Biotechnology Department, Federal

D. Kothari Indian Institute of Technology Guwahati, Guwahati, Assam, India

C. Larroche Blaise Pascal University, Aubière Cedex, France

L.A.J. Letti Bioprocess Engineering and Biotechnology Department, Federal

O. Loera-Corral Universidad Autónoma Metropolitana-Iztapalapa, Mexico City, DF,

A.I. Magalhães, Jr. Bioprocess Engineering and Biotechnology Department,

A.B.P. Medeiros Bioprocess Engineering and Biotechnology Department, Federal

J.D.C. Medina Bioprocess Engineering and Biotechnology Department, Federal

F. Miranda-Hernández Universidad Autónoma Metropolitana-Iztapalapa, Mexico

N.R. Nair CSIR-National Institute for Interdisciplinary Science and Technology

S. Nair Dow Chemicals GmBH, Dubai, UAE

K.M. Nampoothiri CSIR-National Institute for Interdisciplinary Science and