DK146941B - GLUCOAMYLASE AND PROCEDURE FOR PREPARING A HIGH GLUCOSE CONTENT OF SYRUP BY SUCCARIFICATION OF MELTED STARCH - Google Patents

Description

in 146941

The invention relates to a novel glucoamylase and to a process for preparing a high glucose syrup by saccharifying molten starch to glucose.

5 In order to produce glucose industrially from starch today, the saccharification process is mainly used by glucoamylases produced by microorganisms belonging to the genera Rhizopus and Aspergillus. The conditions under which these glucoamylases are used are pH 5.0 and 55 ° C for Rhizopus strain enzymes and pH 4.5 and 60 ° C for Aspergillus strain enzymes.

In addition, the maximum glucose content of the hydrolyzate is about 96% (on a dry matter basis) when these glucoamylases react with the enzyme-melted starch at a concentration of 30%. One reason why glucose yield does not reach 100% is that isomaltose is formed due to a reverse reaction of these glucoamylases. However, a recent report (U.S. Patent No. 3,897,305) has recently published that the reverse reaction of glucoamylases is extremely small in the vicinity of the neutral range and that glucose yield can thus be raised to about 98% when performed. of the reaction at about neutral pH while using pullulanase. The pullulanase has the effect of branching out the starch and increasing the rate of glucamylase action under these near-neutral conditions. With regard to neutral glucoamylases, only one has been reported to date, namely the glucoamylase produced by the waxy-causing fungus CPiricularia oryzae; Kazuo Matsuda et al .; Amylase Symposium, Vol. 9, 1974), but this glucoamylase has low heat stability and thus cannot be used under industrial conditions.

The object of the invention is to provide a glucoamylase which is active at near neutral pH and has sufficient heat stability for use under industrial reaction conditions and which also reacts with starch hydrolyzate to obtain high yields of glucose.

A microorganism strain has been found which produces a novel glucoamylase with optimum activity at a pH of 6.0 - 6.5 and good heat stability.

This glucoamylase is capable of converting a 30 wt% solution of a 10 D.E. (dextrose equivalent) molten starch to a product containing at least about 96% glucose when reacted with the starch hydrolyzate at pH

6.0 - 6.5 at 55 ° C.

Accordingly, the glucoamylase i-r of the invention is peculiar in that it is identical to the glucoamylase formed by the cultivation of strain 15 Stachybotrys subsimplex, FRI 4377.

Thus, the glucoamylase of the invention can be prepared by culturing the Stachybotrys sub-organism strain FRI 4377 in a nutrient medium and extracting the enzyme from the culture fluid.

In the drawing, FIG. 1 shows the relationship between the pH value and the enzyme activity of the enzyme of the invention and the conventional glucoamylases produced by the microorganisms Rhizopus niveus and Aspergillus niger; FIG. 2 shows the relationship between the temperature and the enzyme activity of the enzyme of the invention and the glucoamylase from Piricularia oryzae; FIG. 3 the inactivation curves of the enzyme of the invention when treated at various pH levels; FIG. 4 is a comparison of the enzyme of the invention and the conventional glucoamylases produced by the microorganisms R. niveus, A. niger and P. oryzae, with respect to their relative heat stability.

In the following, the properties of the novel neutral glucoamylase according to the invention are set out in detail and its properties are compared with those of the known glucoamylases.

The term "D.E." is an abbreviation for "dextrose equivalent", and is used to denote a material's content of reducing sugar, calculated as D-glucose and expressed as a percentage of the total dry matter.

The term "starch hydrolyzate" is generally used to denote syrup or a dry product made by partial hydrolysis of starch. Such a product can be prepared by acidic or enzymatic hydrolysis.

The term "molten starch" is used to denote a low starch hydrolyzate. (D.E. from about 2 to about 20).

20 1. Activity and substrate specificity

The enzyme of the invention is capable of hydrolyzing such carbohydrate compounds as starch, soluble starch, amylose, amylopectin and glycogen, and to generate dextrose therefrom. The yield of dextrose from each of these 25 substrates is 100% when the substrate concentration is 1%. The mutarotation of the dextrose produced is positive.

Thus, this enzyme is a glucoamylase. The reaction rate of the enzyme was compared with the rates exhibited by the glucoamylases produced by Rhizopus and Aspergillus microorganisms in association with various substrates. The results are set out in Table I. As can be seen from this table, the activity of the enzyme of the invention is noticeably higher than the activities of the other two glucoamylases, especially in the hydrolysis of pullulan.

TABLE I

Substrate Specificity a) Reaction Rate_

Enzyme Asperaillus niger Rhizopus

Substrate According to,, b) Level Up. Slug * n ^ lase ,,, ^ 5imylase1 Dextrin (D.E. 10) 100 100 100

Amylopectin 104 113 91

Soluble starch 122 95 112

Pullulan 9 2 2

Glycogen 102 100 91 15 Maltotriose 6 12 8

Maltohexose 91 100 146

Panose 44 47 48 .Maltose 14 26 19 a) the enzymatic activities of each glucoamylase were determined with the substrates present at a concentration of 1%; each enzyme's activity in relation to dextrin was assigned the value 100, and the activities on the other substrates are given as relative values.

b) Negotiated by Enzyme Development Corporation, 2 Penn 25 Plaza, New York, N.Y.

"Sumyzyme" negotiated by Sumitomo Shoji Kaisha, Ltd., 1, Kanda Mitoshiro-Cho, Chiyoda-ku, Tokyo, Japan.

5 146941 2. pH optimum and stable pH range

The dependence between the enzymatic activity (relative value) of the enzyme of the invention and the pH of the reaction was investigated and then compared with the corresponding 5 dependencies for the conventional known glucoamylases produced by the Rhizopus and Aspergillus microorganisms. The results are shown in FIG. 1. As shown in the figure, the pH optimum for this enzyme at 60 ° C is 6.0 - 6.5, which is significantly higher than the pH optimum for the other enzymes.

Furthermore, this enzyme shows its best stability in the vicinity of pH 6.0, but no inactivation of this glucoamylase over pH range 4-11 is seen, even when left on the substrate for 24 hours at room temperature.

3. Strength Assay 15 A 0.5 ml aliquot of a suitably diluted enzyme solution was added to 0.5 ml of a 2% solution of a spray-dried maltodextrin (DE about 10) in 0.1 M acetate buffer solution (pH 6.0), and the mixture was incubated at 60 ° C for exactly 10 minutes. The enzyme reaction was then quenched by heating the mixture for 5 minutes in a boiling water bath. The amount of dextrose produced was determined by the glucose oxidase method. The amount of enzyme that produced one micromole of dextrose per minute was defined as 1 unit.

25 4. Temperature range for optimal reaction

The temperature effect on the relative enzymatic activity of the enzyme of the invention at pH 6.0 was compared with the relative activity of the known glucoamylase from the rice welding fungus, Piricularia oryzae. This comparison is shown in FIG. 2. It is clear that, under these conditions, the optimum temperature for the reaction of the enzyme of the invention is 65 ° C, ca. 10 ° C higher 6 146941 than the optimal temperature for the enzyme from Piricularia oryzae, 5. Inactivation due to pH and temperature conditions

FIG. 3 shows inactivation curves for the relative enzymatic activity of the enzyme of the invention when treated for 60 minutes at 60 ° C and over a pH range of 3- 8. As seen in the figure, this enzyme is most stable at pH 6, and it is completely inactivated by this treatment for 30 minutes at pH 3 and for 1 hour at pH 10 4. In addition, FIG. 4 is a comparison of the heat stability of the enzyme of the invention and the glucoamylases of the Rhizopus, Aspergillus and Piricularia microorganisms. FIG. 4 provides inactivation curves for these enzymes when treated at 60 ° C while maintained at their respective pH optima for stability. It can be seen that the heat stability of the enzyme of the invention is inferior to the heat stability of the glucoamylase from Aspergillus, but is better than the heat stability of the glucoamylases of Rhizopus and Piricularia microorganisms.

20 6. Inhibition, Activation and Stabilization

The enzyme of the invention requires no special activating or stabilizing agents. However, as is the case for most other glucoamylases, this enzyme is inhibited by mercury (II) chloride, potassium manganate, iron (II) -25 chloride, other metal salts and "tris".

7. Purification procedure

The enzyme of the invention can be purified by a combination of any of the general purification methods such as ammonium sulfate fractionation, organic solvent fractionation, starch adsorption and various chromatography. An illustrative example of such a cleaning procedure is given below.

7 146941

The cells and other insoluble material are removed from the culture material and the culture liquid is frozen overnight at -20 ° C. It is then melted at room temperature and the insoluble material is removed by centrifugation. Then 2 volumes of cold isopropanol are added and the mixture is allowed to stand overnight at 4 ° C. The enzyme precipitate is freed from the supernatant by decantation. The precipitate is then dissolved in a 0.05 M tris / HCl buffer solution (pH 7.5) containing I.jdM EDTA, and the dissolved material is then dialyzed overnight at 4 ° C over the same buffer. Then DEAE cellulose which has been equilibrated with the same buffer solution is added to this dialyzed enzyme solution so that the enzyme is adsorbed thereto. After washing this DEAE cellulose with the same buffer, the enzyme is eluted from the resin with a preparation of the same buffer containing 0.3 NaCl. The enzyme is then precipitated by adding 2 volumes of cold isopropanol to the eluate and the precipitated material is recovered by centrifugation.

The precipitate is dissolved in the 0.05 M tris / HCl buffer solution 20 (pH 7.5) containing 1 mM EDTA followed by overnight dialysis against the same buffer. The dialyzed enzyme solution is then loaded onto a DEAE cellulose column which has been equilibrated with the same 0.05 M tris / HCl buffer (pH 7.5) containing 1 mM edta. The enzyme is eluted from this column by a linear concentration gradient of the same buffer containing NaCl up to 0.5 M.

The eluted fractions containing the enzyme are pooled and the enzyme is concentrated by the isopropanol precipitation technique. This concentrated enzyme is then loaded onto a column of cross-linked dextrangel "Sephadej G-150" which has been equilibrated with a 0.05 M tris / HCl buffer (pH 7.0) containing 1 mM EDTA and the elution is performed with the same buffer solution. Following this procedure, the purified enzyme showed a single band by disk electrophoresis.

8. Molecular Weight

The molecular weight of the enzyme of the invention was tested using a "Sephadex (¾ -150") column in accordance with the procedure described by Dunker / AK and Rueckert, RRJ Biol. Chem. 244, 5074 (1969 The results showed that the molecular weight of this enzyme 5 is about 50,000.

The following explains the differences between the enzyme of the invention and the conventionally known glucoamylases and the reasons why this enzyme must be considered a novel enzyme with its pH optimum near neutrality.

10 It can be seen from the 1 and Table II, the data indicated that the only glucoamylases having their pH optima close to the neutral zone are the enzyme of the invention and the glucoamylase produced by the rice widening microorganism, Piricularia oryzae. However, as shown in FIG. 2 and Table II, that the enzyme of the invention and the rice widening glucoamylase have extremely different optimum reaction temperatures. In addition, they show in FIG. 4 shows that the heat stability of the enzyme according to the invention is far better than that of the rice welding glucoamylase. Furthermore, Table II shows that the molecular weight of the enzyme of the invention is much less than the molecular weight of the known glucoamylases.

TABLE II

Comparison of various glucoamylases with respect to 25. pH optimum, optimum temperature and molecular weight 9 146941 pH optimum temperature Molecular weight3

a) optimum ° C

Glucoamylase _ a) _ _

Enzyme of the Invention (Stachybotry's Subirplex) 6.0-6.5 * 65X 50,000 *

Rhizopus sp. ("Sumyzyme") 5.0X 60x 70 000 * 5 ^ 5 Aspergillus niger 4.5X 70x 97 000 °

Endcmyces sp. 5.0 - 64,000 p)

Endomyces fibuligera 5.5 60

Trichoderma viride ^ 5.0 60 75 000

Cephalosporium 10 charticolag ^ 5.4 60 69 000 U \

Piricularia oryzae '6.5 55 94 000 (Scratch welding org.) A) All values except those marked with an asterisk (x) are taken from the following references: 15 b) Hiromi et al .: Biochem. Biophys. Acta 302, (1973).

c) J.H. Pazur et al .: J. Biol. Chem. 237, 1002 (1962).

d) Hattori et al .: Agr. Biol. Chem. 25, 895 (1061).

e) Harada et al .: J. Ferment. Tech. 53, 559 (1975).

f) Okada; J. Jap. Soc. Starch Sci. 21, 283 (1974).

20 g) h. Urbanek et al.: Appl. Micro. 30, 163 (1975).

h) Matsuda et al .: Amylase Symposium, 105 (1974).

On the basis of the above data, it can be concluded that the glucoamylase of the invention is a new neutral glucoamylase which has been unknown to date.

The following explanation is intended to illustrate in more detail the process of producing the enzyme of the invention.

The glucoamylase-producing microorganism, strain G. 30-1140, FRI 4377, was isolated from the soil by the inventors in this case. The identification of this strain must first be indicated. The morphological characteristics of the strain were determined according to the methods described by the researchers listed below: ίο 146941

Glimen, J. C. A MANUAL OF SOIL FUNGI. The Iowa State Uni- versity Press, Ames. 1,971th

Clements, F. E. and Shear, C. L. THE GENERA OF FUNGI.

Down, New York. 1964th

5 Barnett, H. L. ILLUSTRATED GENERA OF IMPERFECT FUNGI.

2nd ed. Burgess, Minneapolis. 1968th

Bisby, G. R. Trans. Br. Myco. Soc. 26, 133-43 (1943).

Ainsworth, G. C. DICTIONARY OF THE FUNGI. 6th ed. Common-wealth Mycological Institute, Kew, Surrey. 1,971th

10 9. Morphological properties of strain G30-1140

The strain was grown on 5 kinds of media in Petri bowls. The following sections indicate the morphological characteristics observed for isolated colonies.

a) Czapek agar medium 15 After incubation at 30 ° C for ten days, the colonies are thin and round with a diameter of 4-5 cm. The vegetative hyphae are clear and show little growth with black conidia clusters scattered like powder over the colonies. The undersides of the colonies are brown and a light brown pigment is excreted to the medium.

The vegetative hyphae consist of branched fibers, which rarely have some septa; the conidia structure is uniformly supported by the fibers. The conidiophores, which have septa, protrude from this at right angles. The length 25 of the conidiophores is usually 40-60 µm, but sometimes reaches more than 100 µm. The diameter of these is from about 3 to about 6 µm, and although there are cases where their basic area is smooth, most of their surface is wart covered with fine granular projections. These conidiophores are glass clear and most are not branched.

At the tip of the conidiophores, clear glass phialids form 5 twists of 3-8 units. The shape of the phialides is egg-shaped or flask-like; they are 8-15 µm long and have a diameter of 2-6 µm; their surface is smooth. The conidia form at the tip of the phialides and are oval 3-5 µm x 5-10 yum and have a smooth surface. These are crystal clear at the time-10 point of their formation but become black-green as they mature.

The surface of the conidia is covered with a large amount of viscous material. For this reason, the conidia cleave together and form large conidia clusters at the tip of the conidiophores. The viscous material is transparent at the time of formation but then gradually becomes black.

b) Modified Czapek agar medium

Percentage

Soluble Starch 1.0

Maize casting liquid (dry matter basis) 0.1 NaNO ^ 0.2 k2hpo4 0.1 KCl 0.05

MgSO4'7H20 0.05

FeSO 4 * 7H 2 O 0.001 Agar 2.0

pH to 0.7 with NaOH

The growth of the colonies on this medium is slightly slower than on the Czapek medium described above, reaching about 3 cm when incubated for 10 days at 30 ° C.

12 146941

The colonies are circular and thin, and their surfaces radiate from their centers a black viscous material which is in the form of oil-like drops with diameters reaching 1-3 mm. These arise from the fusion of 5 conidia clusters, which are enclosed in the viscous material, thereby forming oil droplet-like bodies. The undersides of the colonies show a darker brown color than seen with the above Czapek medium, and a small amount of brown pigment is excreted into the medium.

10. c) Potato dextrose agar medium

The growth of the colonies on this medium is slightly slower than on the Czapek medium described above, reaching 3-4 cm when incubated for 10 days at 30 ° C.

These colonies are also circular, but they have a somewhat greater thickness than the colonies on the Czapek medium. The growth of the vegetative cells is good and it develops in a radiation pattern. The colonies' surfaces are black with a faint green tinge and are rich in hypher, conidia clusters, etc.

After 14 days of incubation, the surfaces of the ancient colonies have radiating formations of synnemata standing about 1-3 mm upright. The undersides of these colonies show a blackish brown color and a large amount of brown pigment is excreted to the medium.

d) Special Agar Medium 25 Percent

Soluble Starch 1.0

Corn molding liquid (dry matter basis) 0.2

Cotton seed oil residue 0.1 Yeast extract 0.1 K2HP04 0.1

MgSO4 * 7H2D 0.05

Agar 2.0

pH to 7.0 with NaOH

146941 13

The colonies of this medium, after 10 days of incubation at 30 ° C, have diameters of 5-6 cm and are round and thin. The vegetative hyphae show good growth and have a black tinge. Co-nidia growth is poorer than on the aforementioned media.

5 The undersides of the colonies are light brown and a light brown pigment is released to the medium.

e) Detect yeast-salt-agar medium

The colonies of this medium, after 10 days of incubation at 30 ° C, have diameters of 2-3 cm and are more oval than round 10 of shape. The hyphae are light brown with a tinge of white and form somewhat thick colonies which are velvety. The undersides of the colonies are light brown, but no pigment is secreted into the medium.

10. Physiological properties of strain G30-1140 15 a) Growth temperature

This strain is capable of growing over a temperature range of 10-37 ° C, but its optimum growth temperature is near 30 ° C.

b) Growth pH

This strain is capable of growing over a pH range of 3 - 10, but its optimum growth pH is in the vicinity of pH 7.

c) Carbon sources

This strain is capable of utilizing such carbon sources as dextrose, fructose, galactose, mannose, saccha rose, maltose and starch to support growth.

14 146941 Based on the above microbiological findings, strain 630-1140 was identified as Gliobotry's albovirides after posting in the genera of fungi and a manual of SOIL FUNGI. However, according to DICTI0__ 5 NARY OF FUNGI and G.R. Bisby (Trans. Br. Mycol. Soc. 26, 133-43 (1943) the same as Stachybotry's subsimplex, and for this reason strain G30-1140 was identified as Stachybotry's subsimplex).

Strain G30-1140 has conidiophores that arise from its 10 vegetative hypers, branches hardly exist and they have s.epta. There are cases where the base is smooth but the tip is covered with protrusions. At the apex, a level of phialids forms a spiral of 3-8 units. Conidia with smooth oblong surfaces divide from these phia-15 sufferers and are enclosed in a rich viscous substance. These properties are in good agreement with those described for Stachybotry's subsimplex by G. R. Bisby (Trans.

Br. Mycol. Soc. 26, 133-43 (1943)).

This Stachybotry subsimplex strain G30-1140 is deposited 20 in The Fermentation Research Institute, Agency of Industrial

Science & Technology, Chiba City, Japan under No. 4377.

With regard to the cultivation of this microorganism, the general knowledge and technique in the cultivation of fungi can be used.

25 As a nutrient medium it is possible to use the media used for the cultivation of ordinary mushrooms. For example, various starches, starch hydrolysates, corn flour, wheat flour, molasses, etc. can be used as carbon sources, while the nitrogen requirements can be met in the form of peptone, cotton 3Q seed oil residues, meat extract, yeast extract, casein, corn dust liquid, malt extract, soybean residues, skimmed milk, inorganic ammonium salts, inorganic nitrates, etc. As in organic salts, it is possible to use calcium chloride, magnesium sulfate, phosphates, sodium chloride, potassium chloride, etc. Furthermore, these carbon sources, nitrogen sources and inorganic salts can be used either suitable combinations. In addition, if one wishes to promote the growth of the microorganism and produce an increase in its enzyme production, it is possible to use trace amounts of metal salts, vitamins, amino acids, etc.

The cultivation conditions usually used for 10 fungi can also be used in the cultivation of this micro organism. If the microorganism is grown in liquid culture for 7-14 days at pH 5 - 8 and at 20 - 37 ° C and with stirring to give aeration, the enzyme of the invention accumulates in the culture liquid. If solid materials such as bran are used, it is also possible to perform solid cultivation.

The following is an example of a method by which the novel neutral glucoamylase of the invention can be recovered from the culture material. In the case of liquid culture, the mycelium is removed by any of the generally known methods. Then, the filtrate can be concentrated under reduced pressure, or the enzyme can be salted out with the other proteins by addition of inorganic salts, such as ammonium sulfate, to the filtrate, or the enzyme can be precipitated and concentrated by the addition of an organic solvent such as acetone or isopropanol. .

In the case of a solid culture, the enzyme is first extracted from the culture material using water or a buffer solution. Then, as in the liquid culture, it is possible to obtain the enzyme in concentrated form.

The crude compositions thus obtained can then be purified by carrying out the aforementioned purification technique.

16 146941

It is possible to use the glucoamylase of the invention for the saccharification of molten starch to produce dextrose from starch. In particular, when using the enzyme of the invention for saccharification at a pH of 6.0 - 6.5, as previously mentioned, only a slight reverse reaction occurs, and this results in an increased yield of dextrose in comparison with what is is the case using the conventional glucoamylases and performing the saccharification under acidic conditions.

Accordingly, the process of preparing a high glucose syrup by saccharifying molten starch to glucose is peculiar in that the molten starch is saccharified at a pH of 6.0 - 6.5 in the presence of the glucoamylase of the invention.

The saccharification is preferably carried out at a temperature of from about 50 to about 65 ° C.

The invention is further illustrated by the following examples, in which all parts and percentages are by weight, unless otherwise stated.

EXAMPLE 1

A liquid culture medium containing 5% soluble starch, 2% maize casting liquid, 0.5% cotton seed oil residue, 0.5% yeast extract, 0.1% dicalcium phosphate, 0.05% magnesium sulfate and 0.01% calcium chloride was adjusted to pH 7 , 0, and 100 ml of this medium were placed in a 500 ml Erlenmeyer flask. The medium was sterilized at 121 °. C for 10 minutes, seeded with Stachybotry's subsimplex strain G30-1140, FRI 4377, and incubated at 30 ° C for 7 days on 30 shakes. After completion of the culture, the mycelium was removed from the culture fluid by filtration. The filtrate was found to contain 70 units of glucoamylase activity per ml. ml.

17 146941

This filtrate was frozen overnight at -20 ° C and then thawed at room temperature. The insoluble material was removed by centrifugation. Then 2 volumes of cold isopropanol were added to this solution and allowed to stand at 4 ° C for one night so that the enzyme precipitated. The supernatant was removed by decantation and the precipitate dissolved in a 0.05 M tris-HCl buffer solution containing 1 mM EDTA and with a pH of 7 »5. This enzyme-containing solution was then dialyzed against the same buffer at 4 ° C overnight. DEÅE cellulose which had been equilibrated with the same buffer solution was then added to the dialyzed enzyme solution and the enzyme was adsorbed to this carrier. After washing the DEAE cellulose with the same buffer, the enzyme was eluted from it using a solution of the same buffer containing NaCl at a concentration of 0.3 M.

Then, two volumes of cold isopropanol were added to the eluate to precipitate the enzyme and the precipitate was collected by centrifugation. This precipitate was dissolved in 0.05 M tris / HCl-20 buffer (pH 7.5) containing 1 mM EDTA, followed by overnight dialysis against the same buffer solution.

The dialyzed enzyme solution was loaded onto a column of DEAE cellulose which had been equilibrated with the same 0.05 M tris / HCl buffer (pH 7.5) containing 1 mM EDTA.

Elution of the enzyme from this column was performed by linearly increasing the concentration of NaCl in the same buffer solution up to 0.5 M. The fractions of the eluate containing the enzyme were then pooled and two volumes of cold isopropanol were added to to precipitate the enzyme from this solution and concentrate it. The concentrated enzyme was then loaded onto a column of "Sephadex ™ G-150", which had been equilibrated with a 0.05 M tris / HCl buffer solution (pH 7.0) containing 1 mM ΕΚΤΑ, and eluted with the same buffer. The eluted fractions showing enzyme activity were then pooled and two volumes of cold isopropanol were added to precipitate the enzyme. This resulted in the recovery of the enzyme in purified and concentrated form. The specific activity of the purified enzyme was found to be 127 units per mg of protein.

EXAMPLE 2 To a 30% solution of a spray-dried maltodextrin (D.E. about 10) in 0.05 M acetate buffer at pH 6.5, the purified glucoamylase of Example 1. The enzyme was added at a dosage of 0.20 units of enzyme per ml. g of dry matter substrate. After the solution was incubated at 55 ° C for 72 hours, the dextrose content of the filtered hydrolyz, as determined by high performance liquid chromatography, was 96.5% of the total carbohydrate.

EXAMPLE 5

Starch was transformed into a 10.2 D.E. starch hydrolyzed using bacterial α-amylase from B. licheni formis according to the general procedure used in U.S. Patent No. 3,912,590. The solution was boiled for 5 minutes after adjusting the pH to 2.0 . with 2 NHCl to inactivate the residual α-amylase. The starch hydrolyzate solution was then adjusted to pH 6.2 and diluted to the desired concentration before treatment with 0.20 units of the purified glucoamylase of Example 1 per ml. g substrate (dry matter basis). The solution was incubated at 55 ° C in a plugged test tube. The pH was adjusted to 6.2 after 5 hours and after 48 hours. After the solution was incubated for 2 hours, the dextrose content of the filtered hydrolyzate, as determined by high performance liquid chromatography, was 97.6% of the total hydrocarbon. The final concentration of the solution was 31.2% on a dry matter basis.

When saccharification tests were performed at the same substrate concentration with A. niger commercial glucoamylase 5 under its optimum conditions (pH 4.3 at 60 ° C), the corresponding dextrose yield was 96.5%. Similarly, the glucoamylase from R. levels at pH 5.0 and 55 ° C gave a dextr ose yield of 97%. The dextrase yields were about 1% lower when the saccharification tests were performed with the commercial glucoamylases under the conditions used for the enzyme of the invention. These results show that the novel glucoamylase of the invention provides higher yields of dextrose than the commercial glucoamy laser, even when each enzyme is used under its optimal reaction conditions.