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Feature papers are submitted upon individual invitation or recommendation by the scientific editors
and must receive. Lane M—molecular mass marker in kDa, lanes 1 and 2—crude extract, lanes 3 and
4—partially purified crude extract, and lanes 5 and 6—concentrated supernatant. Other product and
company names shown may be trademarks of their respective owners. Resource includes a starter
question, video, method sheet, questions and answers. Journal of Otorhinolaryngology, Hearing and
Balance Medicine (JOHBM). Get this resource as part of a bundle and save up to 48% A bundle is a
package of resources grouped together to teach a particular topic, or a series of lessons, in one place.
N utrition - energy, growth and repair, eg: proteins, fats, carbohydrates, vitamins, minerals 5.
Sequences of the primers employed for the amylase encoding genes amplification. To make sure that
accurate results are gained repeat the experiment two to three times. In addition to temperature, pH,
and concentration other factors such as the structure of the substrate, the amount of concentration of
the substrate, the ionic strength of the solution, and the presence of other molecules that can act as
activators or inhibitors 1.For the factors that were examined, it was predicted that as amylase
concentration decreases the enzyme function decreases as measured by the rate of starch digestion.
Course Outcome. Ability to discuss the technologies available in bioconversion. HS, the haloarchaea
was cultured in a (i) rich medium, which contained yeast extract and a (ii) minimal medium, in which
the yeast extract was substituted by ammonium acetate. Furthermore, both amylase activities
showed high stability in most tested surfactants, excepting the anionic detergent SDS. Safety is very
important when doing experiments like this. What are the characteristics of each type of reactor.
Figure S3: Predicted structures of the three amylase sequences identified in Haloarcula sp. HS.
Figure S4: Multiple alignments of the amino acid sequence of the extracellular amylase identified in
Haloarcula sp. This is because of the limitation of the number of starch molecules available. This
article is an open access article distributed under the terms and conditions of the Creative Commons
Attribution (CC BY) license ( ). You can see in the table below how this will be done to achieve the
amount I need. The whole gels for each staining are available in Supplementary Material, Figure S2.
HS, exhibit a strong inhibition in the presence of the metal chelating agent EDTA ( Figure 6 ).
Enzymes therefore work efficiently at very low concentrations. The red arrow indicates the moment
of transference of the cells from the rich to the fresh minimum medium. I did this by going through
each concentration of amylase and washed them out before each concentration. An assay is usually
performed to determine this type of information. It would not be likely to have a great impact on my
results as water is able to retain heat well therefore resulting in the error being minor. As shown in
Figure 2, cell growth and protein secretion were higher when the microorganism was grown in the
rich media, which contained yeast extract. Since the minimal medium had no yeast extract, there are
no foreign proteins that could interfere with the amylase secreted into the culture medium. Any
spillage of iodine or starch will be washed thoroughly with soap and water. The red arrow indicates
the moment of transference of the cells from the rich to the fresh minimum medium.
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Therefore, intra- and extracellular proteins from haloarchaea are specially adapted to work properly
at high salt concentrations. Information cookies Cookies are short reports that are sent and stored on
the hard drive of the user's computer through your browser when it connects to a web. Afterwards,
the produced quantity of reducing sugars released from starch is determined as described previously.
If you want you can disable cookies from Google Analytics. The rate of a reaction depends on more
factors than the three examined. Extracellular and cell-associated relative amylase activities under
the presence of different metal ions and EDTA (10 mM) are represented. Therefore, more insightful
biochemical characterization studies are needed to reveal the exact features of these amylolytic
enzymes from haloarchaea. HS Isolated in the Odiel Marshlands. Biology. 2021; 10(4):337. In the
first trial (figure 2) the activity of amylase (as measured by time taken to fully digest starch) had no
logical correlation as the serial dilutions decreased in concentration of amylase. To our knowledge,
this study supposes the first attempt to use a halophilic amylase to degrade starch from bread into
simple sugars. The tree represents a comparison among the complete 16S rRNA coding gene
sequences, including a series of reference haloarchaeal species and the new isolated strain,
Haloarcula sp. HS. Multiple alignments were generated by MUSCLE (MUltiple Sequence
Comparison by Log-Expectation) and the tree was constructed with MEGA X. It also gives me an
insight to how many times to repeat the experiment. It is appropriate for both standard and honors
classes as well as for first or second year biology students. In addition, the introduction and abstract
are sometimes merged into one section in a college setting. Title. Journal of Low Power Electronics
and Applications (JLPEA). The amylase activity was induced by the addition of starch (3 g L ?1 ) to
either the rich or the minimal medium. 2.4. Partial Purification of Cell-Associated and Extracellular
Amylases Haloarcula sp. A standard curve is being drawn Then the absorbance of each one of the
unknown samples is measured and the concentration of the converting sugars is determined, based
on the standard curve. Therefore, a two-step culture was set up, in which a large amount of biomass
was obtained in a rich medium, followed by transfer to a minimal medium with starch, to induce the
production of amylase activity ( Figure 3 ). Provided the temperature and other conditions are
suitable for the reaction, the rate of a reaction should be directly proportional to the enzyme
concentration’ (page 35). This lab is designed for a typical high school biology class for students in
grades 9 - 12. In order to be human-readable, please install an RSS reader. S ensitivity - reacting to
changes in their surroundings 4. Proteases include Pepsin and Trypsin, which help to digest proteins,
and Chymotrypsin, which help to digest Polypeptides. Optimal parameters reported for ?-amylase
activity in haloarchaea. The PCR products were analyzed by electrophoresis on a 1% agarose gel and
sent to Stabvida (Lisbon, Portugal) for Sanger sequencing. Finally start the stopwatch as soon as the
two substances have been mixed, and stop it straight as the colour has gone. The tree represents a
comparison among the complete 16S rRNA coding gene sequences, including a series of reference
haloarchaeal species and the new isolated strain, Haloarcula sp. HS. Multiple alignments were
generated by MUSCLE (MUltiple Sequence Comparison by Log-Expectation) and the tree was
constructed with MEGA X. The amount of remaining starch was measured by the iodine-starch
method. In addition, amylases are widely used in textile and paper industries to remove the starch
employed for the desizing process and the coating treatment, respectively.
These proteins are produced by a wide variety of organisms and are supposed to be one of the most
valuable industrial enzymes. Safety is very important when doing experiments like this.
Carbohydrases include Amylase, which helps to digest starch, Lactase, which helps to digest
Lactose, Sucrase, which helps to digest Sucrose, and Maltase, which helps to digest Maltose. HS
cells were first cultured in the rich medium, containing yeast extract and starch. Lane M—molecular
mass marker in kDa, lanes 1 and 2—crude extract, lanes 3 and 4—partially purified crude extract,
and lanes 5 and 6—concentrated supernatant. This two-stage method allowed the improvement of
amylase production, and at the same time facilitated the recovery of the extracellular enzyme. This
fact contributes to broadening the range of optimal temperatures for the cell-associated amylase
activity. The obtained sequences were analyzed using the CLC Workbench software (version 8,
Qiagen, Hilden, Germany). 2.9. Identification of Amylase Coding Genes Based on Protein
Sequences With the aim of completing the full protein sequences of the amylases identified, the
sequences of their encoding genes were amplified by PCR, using sets of primers specifically
designed on the basis of the sequences of peptides obtained in the proteomic analysis. Therefore, a
two-step culture was set up, in which a large amount of biomass was obtained in a rich medium,
followed by transfer to a minimal medium with starch, to induce the production of amylase activity (
Figure 3 ). Furthermore, both amylase activities showed high stability in most tested surfactants,
excepting the anionic detergent SDS. Write a review Update existing review Submit review Cancel
It's good to leave some feedback. Overall, the results suggest that enzyme activity may be affected
by factors such as concentration, pH, and temperature. The Lab Sheet (exercise) should be filled and
signed by the teaching engineer at the end of each lab session. Bundle AQA GCSE BIology Trilogy
Topic 2: Organisation bundle All lessons included plus practical sheets ?30.00 Reviews Select
overall rating (no rating) Your rating is required to reflect your happiness. Journal of
Otorhinolaryngology, Hearing and Balance Medicine (JOHBM). Our customer service team will
review your report and will be in touch. ?2.00 (no rating) 0 reviews BUY NOW Save for later ?2.00
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through email Share through twitter Share through linkedin Share through facebook Share through
pinterest BSP Biology 1.67 3 reviews Not the right resource. As the temperature of the reaction
moves past its optima, although chemically the chances of starch breakdown increase, the chances of
the breakdown of the three-dimensional structure of the enzyme also increase. Chapter 5
Considerations in Using Plant and Animal Cell Cultures. A control, containing starch recovered from
bread without the enzyme solution, was incubated in the same conditions. I will dilute the solution of
amylase by adding water to it. Reviews Select overall rating (no rating) Your rating is required to
reflect your happiness. Once formed, the enzyme may leave the cell and work outside. Also included
is a 4-page handout for the instructor that contains teaching tips and suggestions, information for
how to prepare the solutions, lab set-up tips, and answers to all questions. The rate of a reaction
depends on more factors than the three examined. Investigating factors which affect the rate of the
Amylase Enzyme in conver. HS, the haloarchaea was cultured in a (i) rich medium, which contained
yeast extract and a (ii) minimal medium, in which the yeast extract was substituted by ammonium
acetate. Purple stars highlight the catalytic triad (Asp-Glu-Asp), blue star denotes the canonical
calcium-binding site and the black stars point other essential residues for enzyme structure. HS or
due to the contribution of additional cell-associated enzymes. Furthermore, the potential applicability
of the amylase enzymes of this new haloarchaea on the treatment of bakery waste was assessed and
compared with a commercial amylase. 2. Materials and Methods 2.1. Screening and Selection of
Amylase Producing Haloarchaea The screening of amylase-producing haloarchaea was performed by
detection of the extracellular amylase activity of the isolates on starch agar plates, with 20% NaCl.
The rate of reaction will be slower if a lower concentration of amylase solution is used; this is due to
the fact that there would be a competition for the substrate to react with the active sites. Therefore to
gain accurate readings I read off from the bottom of the curve. At 30 seconds it could have been 24
or at 1min 30sec it could have been 1min24sec. International Journal of Translational Medicine
(IJTM). HS cells were first cultured in the rich medium, containing yeast extract and starch. Relative
activity was defined as the percentage of maximum activity for each case. Phyre2 software and the
Chimera program were employed for 3D structure visualization. Maltose is the main end-product
released from the starch hydrolysis by the extracellular and cellular partially purified amylase extracts
of Haloarcula sp. In order to be human-readable, please install an RSS reader. Instead of using 13
test tubes at the same time I used only two. These kinds of chemical identifications of enzyme
substrate and products are called enzyme assays. Amylases are a diverse group of hydrolase or
transferase enzymes that degrade large alpha-linked polysaccharides, such as starch and related
oligosaccharides, and are one of the most required enzymes in industrial operations. The lab report
for each session should be submitted within ONE week after the experiment was performed. This
should be the longest section and: Explain what the results mean. The obtained hydrolysate was very
rich in simple sugars that could now be used for the bioproduction of many high-value molecules
like glycerol, hydrogen, ethanol, or lactic acid, among others. The need for poly-extremotolerant
amylases encouraged their search in extreme environments, where archaea become ideal candidates
to provide new enzymes that are able to work in the harsh conditions demanded in many industrial
applications. In addition, the enzymatic preparation from the cellular extract was also able to catalyze
the liberation of glucose (6.6%). On the other hand, the extracellular extract and the commercial
reference amylase catalyzed the liberation of dextrins, which supposed 18.5% and 20.8% of the total
carbohydrate content in the reaction mixture, respectively, in addition to maltose. Through this
approach, practically the full length of each protein sequence was completed. You are also writing a
hypothesis and telling your reader what that hypothesis is. Prior to trypsin digestion, the samples
were diluted with ammonium bicarbonate 25 mM, until the concentration of trifluoroethanol was
under 5%. The shape of the active site matches the shape of the substrate molecule so the substrate
can fit into the active site and amylase can react and breakdown starch. Discussion The last major
part of writing a lab report is the discussion. Editor’s Choice articles are based on recommendations
by the scientific editors of MDPI journals from around the world. Sometimes acknowledgments are
skipped in less formal reports that are written for a college class. Optimal parameters reported for ?-
amylase activity in haloarchaea. The full length of the 16S rRNA encoding gene was amplified with
the archaeal specific primers 21F (5?-TTCCGGTTGATCCTGCCGGA-3?) and 1492R (5?-
GGTTACCTTGTTACGACTT-3?). A standard curve is being drawn Then the absorbance of each
one of the unknown samples is measured and the concentration of the converting sugars is
determined, based on the standard curve. Analyze all the information to create a conclusion. This
was compared to a commercial thermostable ?-amylase. These results indicate that temperature does
have an effect on amylase’s ability to digest starch.
Next Article in Special Issue Thermococcus bergensis sp. nov., a Novel Hyperthermophilic Starch-
Degrading Archaeon. It would not be likely to have a great impact on my results as water is able to
retain heat well therefore resulting in the error being minor. The amount of iodine used must be
constant as the increase in the amount of starch used can result in the slowing down of the reaction
between amylase and starch. This was repeated for all 5 tubes and the respective times were
recorded. The rate of reaction will be slower if a lower concentration of amylase solution is used;
this is due to the fact that there would be a competition for the substrate to react with the active
sites. They are very useful for supporting student's revision through covering everything they need to
know in the perfect amount of detail, and can be used in a variety of ways. ?5.00 Reviews Select
overall rating (no rating) Your rating is required to reflect your happiness. In addition, amylases are
widely used in textile and paper industries to remove the starch employed for the desizing process
and the coating treatment, respectively. Multiple requests from the same IP address are counted as
one view. HS extracts were able to hydrolyze other glucose polysaccharides, such as carboxymethyl
cellulose, or disaccharides, such as sucrose or lactose, which not contain alpha-1,4-linked glucose.
Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. This
will make sure all the tests are identical which means the experiment will be fair. Mix the two
together and instantly start the stopwatch stir the mixture with a stirring rod but ONLY two full
turns. Our customer service team will review your report and will be in touch. ?2.00 (no rating) 0
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updated 26 May 2021 Share this Share through email Share through twitter Share through linkedin
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resources 4.00 3 reviews Not the right resource. Feature papers are submitted upon individual
invitation or recommendation by the scientific editors and must receive. Biochemical
Characterization of the Amylase Activity from the New Haloarchaeal Strain Haloarcula sp. Also if I
don’t keep the temperature the same throughout the investigation my results wouldn’t be correct
which would mean I wouldn’t be able to draw a fair graph and also some enzymes prefer neutral
conditions, whereas some prefer highly alkaline conditions. Molecular markers (NativeMark TM
Unstained Protein Ladder, Thermo Fisher Scientific, Waltham, MA, USA) were used as a reference
for the molecular weight of proteins. Report this resource to let us know if this resource violates
TPT’s content guidelines. The time taken to digest starch (in seconds) was 50, 50, 20, 10, 20, and 20
respectively. A small proportion of glucose was found in the assay catalyzed by the cell extract. Also
amylase has an optimum shape and will hold it at the optimum temperature. This article is an open
access article distributed under the terms and conditions of the Creative Commons Attribution (CC
BY) license ( ). This point is shown on the graph above, as the line of values increases to a point
where it cannot get any further, therefore continues in a horizontal line as shown. Course Outcome.
Ability to discuss the technologies available in bioconversion. One half was stained with Coomassie
blue and the other with Lugol’s iodine solution to detect both proteins and amylase activity,
respectively. The extracellular amylase activity was selected for this assay, as it works better in high
NaCl concentrations than the cell-associated amylase. A unique band with amylase activity was
observed in the culture medium, after a 100-fold concentration step through ultrafiltration with a 10
kDa cut-off membrane, as indicated in Material and Methods. Make sure that all the apparatus used
are washed, so that there is no solution or dirt, etc, to give inaccurate results. The optical density of
the cultures, protein secretion into the media, and hydrolysis of extracellular starch were followed in
both, rich and minimal medium cultures.