CA1131142A - Glucoamylase from stachybotrys subsimplex - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Process for the production of a glucoamylase having a pH optimum at about 6.0 to 6.5 by a strain of Sachybotrys and the glucoamylase produced thereby.

Description

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BACKGROUND OF THE INVENTION
At present, when producing dextrose indus-trially from starch, the principal glucoamylases employed for the saccharification process are those produced by micro-organism belonging to the genera Rhizopus and Aspergillus.
The conditions under which these glucoamylases are employed are pH 5.0 and 55C for the enzyme of the Rhizopus micro-organism, and pH 4.5 and 60C for the Aspergillus micro-organism's enzyme. In addition, maximum dextrose content of the hydrolyzate is about 96% (dry solids basis) when these glucoamylases react with enzyme liquefied starch at a 30% concentration. One reason that the dextrose yield does not reach 100% is that isomaltose accumulates due to a reverse reaction by these glucoamylases. However, there was recently published a report (U.S. Patent 3,897,305) that the reverse reaction of glucoamylses is extremely small in the vicinity of neutrality and that the dextrose yield can thus be elevated to about 98% by carrying out the reaction at about a neutral pH with the joint use of pullalanase. The pullalanase acts to debranch the starch and increases the rate of glucoamylase action under these nearly neutral conditions. As far as neutral glucoamylases are concerned, only one has ~een reported to date, that being the glucoamylase produced by the rice blast-causing fungus (Piricularia oryzae; Kazuo Matsuda, et al:
Amylase Symposium, Vol. 9, 1974), but t~ls glucoamylase possesses low thermostability and so cannot be employed under industrial conditions.

SUMMARY OF THE INVENTION

It is therefore a primary object of the present invention to provide a glucoamYlase that is active at nearly neutral pH.

It is another object of the invention to provide a glucoamylase that possesses enough thermostability so that it can be employed under industrial reaction conditions.

It is yet another object of the invention to provide a glucoamylase that reacts with a starch hydrolyzate to give high yields of dextrose.

A microbial strain has been discovered which produces a new glucoamylase having optimum activity at a pH of 6.o to 6.5 and good thermostability. The new glucoamylase is capable of converting a 30% by weight solution of a 10 D.E. (dextrose equivalent) liquefied starch to a product containing at least about 96% dextrose when reacted with the starch hydrolyzate at pH 6.0 to 6.5 at 55C. This invention includes the method for the production o~ this glucoamylase wherein the microorganism 11;31142 of the genus Stach,ybotrys, which produces the glucoamy,ase, is cultured in a medium and the enzyme is recovered from ' the culture broth.
.

BRIEF DESCRIPTION OF THE DRA~INGS

Figure 1 shows the relationship between the pH
and the enzyme activity in the cases of the enzyme of the present invention and the conventional glucoamylases produced by _. niveus and A. niger microorganisms.

Figure 2 shows the relationship between the temperature and the enzyme activity in the cases of the present enzyme and the glucoamylase from P. ~

Figure 3 presents the inactivation curves for the enzyme of this invention when it is treated at various 'pH levels.

Figure 4 provides a comparison of the present enzyme and the conventional glucoamylases produced by the R. ni'veus, A. niger and P. oryzae microorganisms in terms of their relative thermostabilities.

DETAILED DESCRIPTION~ OF THE INVENTION

The properties of the novel neutral gluco-amylase of the present invention are presented in detail~
and their properties are contrasted with those of the previously-known glucoamylases.

The term "D.E." is an abbreviation for "dextrose equivalent", and these terms are used interchangeably to refer to the reducing sugar content of a material calculated as dextrose and expressed as percent of total solids.

The term "starch hydrolyzate" is used in a general way to refer to a syrup or dry product that is made by the partial hydrolysis of starch. Such a product may be made by acid or enzymic hydrolysis.

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

1. Activity and Substrate Specificity The present enzyme is able to hydrolyze such carbohydrate compounds as starch, soluble starch, amylose, amylopectin and glycogen, and to produce dextrose from them. The yield of dextrose from each of these substrates is 100% when the substrate concentration is 1%. The muta-rotation of the produced dextrose is positive. This enzyme is thus a glucoamylase. The reaction velocity of this enzyme was compared to the rates shown by the glucoamylases produced by microorganisms belonging to Rhizopus and Aspergillus in relation to various substrates. The results .
are presented in Table I. As can be seen from this table, the activity of the present enzyme is notably higher than the activities of the other two glucoamylases especially in relation to the hydrolysis of pullulan.

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TABLE I

SUBSTRATE SPECIFICITY

; Reaction Ratea) AspergillusRhizopus niger niveus Present Gluco- Gluco-SubstrateEnzymeamylaseb)amylaseC) Dextrin (D.E. 10) 100 100 100 .
Amylpectin 104 113 91 Soluble Starch 122 95 112 Pullulan 9 2 2 Glycogen 102 100 91 Maltotriose 6 12 8 '! Maltohexaose~1 100 146 Panose 44 47 48 Maltose 14 26 19 ' . , , a) The enzymatic activitie$ of each glucoamylase were determined with the sub.strates present at a 1% con-centration; each enzyme t S acti.vity in relation to dextrin was assigned the value 100, and the activities on the other substrates are presented as relative - values.

b) Available from Enzyme Development Corporation,

- 2 Penn Plaza, New York, N.Y.

c) 5umyzyme available from Sumitomo Shoji Kaisha, Ltd., l, Kanda Mitoshiro-Cho, Chiyoda-ku, Tokyo, Japan.

2. Optimum pH and Stable pH Range The relationship between the enzymatic activity (relative value) of the present enzyme and the reaction pH were investigated and then compared with the corre-sponding relationships for the conventionally-known glucoamylases produced by the Rhizopus and Aspergillus microorganisms. The results are presented in Figure 1.

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As shown in the figure, the optlmum pH of this enz~me at 60C is 6.0 to 6.5, considerably higher than the pH optima of the other enzymes. In addition, this enzyme shows its best stability in the vicinity of pH 6.o, but no inacti-vation of this glucoamylase is seen even when it is left sitting for 24 hours at room temperature, over a pH range of 4 to 11.

3. Determination_of Potency 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 (D.E. about 10) in 0.1 M acetate buffer solution (pH 6.o) and this was incubat~ed at 60C for precisely 10 minutes. The enzyme reaction was then stopped 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 producing 1 micromole of dextrose per minute was defined as 1 unit.

4. Optimum Reaction Temperature Range The effect of temperature on the relative enzymatic activity of the present enzyme at pH 6,o was compared with, the relative activity for the knowr. glucoamylase from the rice blast fungus, Piricularia oryzae. This comparison is shown in Figure 2. It is evident that the optimum temperature for the reaction of the present enzyme under these conditions is ~5C, about 10C higher than that of the enzyme from Piricularia oryzae.

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5. Inactivation Due to pH and Tem~erature Conditions .
- Figure 3 presents inactivation curves of the relative enzymatic activity of the present enzyme when it was treated for 60 minutes at 60C over a pH range of 3 to 8. As is clear from 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 4. In addition, Figure 4 shows a comp~rison of the thermostability of the present enzyme and the glucoamylases from the Rhizopus, Aspergillus and Plricularia microorganlsms. Namely, Figure 4 presents the inactivation curves obtained for these enzymes when they were treated at 60C while being held at their respective optimum pH's for stability. It can be seen that the thermostability of the present enzyme is inferior to that of the glucoamylase of Aspergillus - origin, but is superior to the thermostability shown by the glucoamylases from the Rhizopus and the Piricularia micro-organisms.

6. Inhibition, Activation and Stabilization This enzyme does not require any special acti-vating or stabilizing agents. However, the same as in the case of most of the other glucoamylases, this enzyme is inhibited by mercuric chloride, potassium manganate, ferrous chloride, other metal salts and ~ris.

7. Purification Procedure The present enzyme can be purified by means of a combination of any of the ordïnary purification methods ~13,1~

such as ammonium sulfat~ fractianation, organic solvent fractionation, starch adsorption, and various chromatog~
raphies. An lllustrative example ~f such a purification procedure is presented next.

The cells and other insoluble material are eliminated from the cultured material and then the culture fluid is frozen overnight at -20C. This is then melted at room temperature and the insoluble matter is removed by centrifugation. Next, two volumes of cold isopropanol is added to this and it is left standing for one night at 4C. The enzyme precipltates and the supernatant is removed by decantation, The precipitate is then dissolved in a 0.05 M tris-HCl ~uffer solution (pH 7.5) containing 1 mM EDTA, and the dissolved material is next dialyzed for one night at 4C against the same buffer. DEAE-cellulose which has been equilibrated with the same buffer solution is next added to this dialyzed enzyme solution so that the enzyme is adsorbed thereto.
After washing this DEAE-cellulose with the same buffer, enzyme is eluted from the resin with a preparation of the same buffer containing 0.3 M NaCl. The enzyme is then precipitated by the addition of two volumes of cold iso-propanol to the eluate, and this precipitated material is recovered by centrifugation. The precipitate is dissolved in the 0.05 M tris-HCl buffer solution (pH 7.5) containing 1 mM EDTA, followed by overnight dialysis Z
against the same buffer. The dialyzed enzyme solution is ; next applied to 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 then eluted from this column by passing through it a linear concen-tration gradient of the same buffer containing MaCl up to 0.5M. The eluted fractions which contain the enzyme are pooled and the enzyme is concentrated by means of the isopropanol precipitation technique. This concentrated enzyme is then applied to a column of Sephadex* G-150 which has been equilibrated with a 0.05 M tris-HCl buffer (pH 7.0) containing 1 mM EDTA, and elution is carried out with the same buffer solution. When this procedure was followed, the purified enzyme which was obtained showed a single band in disc electrophoresis.

8. Molecular Weight The molecular weight of the present enzyme was investigated using a Sephadex G-150 column in accordance with the procedure of Andrews, P., Biochem J. 96, 595 (1965). The results indicated that this enzyme's molecular weight is about 50,000.
Next, the points of difference between the present enzyme and the conventionally-known glucoamylases will be presented, and an explanation will be made of the reasons that this enzyme is to be considered a new enzyme having its optimum pH in the vicinity of neutrality.

* trade mark .

Regarding the optimum pH.of enzymes, it can be seen from the data presented in Figure 1 and Table II that the only glucoamylases which have their optimum pH's near the neutral zone are the present enzyme and the glucoamylase produced by the rice blast microorganism, Piricularia oryzae. However, as is clear from Figure 2 and Table. II, the present enzyme and the rice blast glucoamylase have optimum reaction temperatures which are extremely different.
In addition, the curves presented in Figure 4 indicate that the thermostability of the present enzyme is vastly superior to that of the rice blast glucoamylase Moreover, Table II
shows that the molecular weight of the present enzyme is much smaller than the molecular weight of the other kno~n glucoamylases.

TABLE II

COMPARISON OF VARIOUS GLUCOAMYLASES IN TERMS
OF OPTIMUM pH, OPTIMUM TEMPERATURE
AND MOLECULAR WEIGHT

Optimum Optimum Temp. Molecular Glucoamylase ~Ha) oCa~ Weighta) Present Enzyme 6.o-6.5 65 50,000 (Stachybotrys subsimplex) Rhizopus sp. (Sumyzyme)5.0 60 70,000b) Aspergillus niger 4.5* 70* 97,000C) Endomyces sp.d) 5.0 - 64,ooo Endomyces fibuligerae) 5.5 60 Trichoderma viride ) 5.0 60 75,000 Cephalosporium charticolag) 5.4 60 69,000 Piricular~a oryzaeh) 6.5 55 94,000 (rice blast org.

a) All values except those marked with an asterisk C~
were taken from the references.
b) Hiromi, et al: Biochem. Biophys. Acta 302, 362 (1973) c) J. H. Pazur, et al: J. Biol. Chem. 237, 1002 (1962).
d) Hattori, et al: Agr. Biol. Chem. 25 ~ 95 ~1961).
e) Harada, et al: J. Ferment. Tech. 53, 559 (1975).
f) Okada; J. Jap. Soc. Starch Sci. 21, 283 (1974).
g) H. Urbanek, et al: Appl. Micro. 30, 163 (1975).
h) Matsuda, et al: Amylase Symposium 2, 105 (1974).

On the basis of the above facts, it can be con-cluded that the glucoamylase produced by the method of the present invention is a new neutral glucoamylase which has been totally unknown to date.

An explanation will now be made of the method for the production of the present enzyme.

As a desirahle example of the glucoamylase-producing microorganism to be used in the present inven-tion, there is strain G30-1140, which was isolated from the soil by the present inventors. The identification of this strain will be presented first.

T~emorphological properties of tne present strain were determined in accordance with the methods described by the researchers listed below:

Gilman, J. C. A MANUAL OF SOIL FUNGI. The Iowa State University press, Ames. 1971.
Clements, F. E. and Shear, C. L. THE GENERA OF
FUNGI. Hafner, New York. 1964.
Barnett, H. L. ILLUSTRATED GENERA OF IMPERFECT
FUNGI. 2nd ed. Burgess, Minneapolis. 1968.

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3isby, G. R. Trans. Br l~lycol. Soc. 26, 133-43 (1943).
Ainsworth, G. C. DICTIONARY OF THE FUNGI.
6th ed. Commonwealth Mycological Institute, Kew, Surrey. 1971.

9. Morphological Properties of Strain-G30-1140 The present strain was cultured on five kinds of media in Petri dishes. The following sections present the morphological characteristics which were observed for isolated colonies.

a) Czapek Agar Medium When incubated at 30C for 10 days, the colonies are thin and round, with a diameter of 4 to 5 cm. The vegetative hyphal are hyalin and show poor growth with hlack conidial clusters scattered like powder over the surface of the colonies. T~e undersides of the colonies are a brown color, and a tan pigment is secreted into the medium.

The vegetative hyphal consist of branched fibers which rarely possess any septa; the conidial structure is uniformly supported by the fibers. The conidiophores which have septa protrude from this at right angles. The length of the conidiophores is usually from 40 to 60 ~, but sometimes they attain more than 100 ~.
The diameter of these is about 3 to 6 ~, and although there are cases when the basal area of these is smooth, most of their surface is verrucose, being covered with fine granular projections. These conidiophores are hyalin, and most are not branched.

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On the apex of the cGnidiophores, hyalin phialides form whorls of 3 to 8 units. The shape of the ph~alides is ovoid or flask-like; they are 8 to 15 ~
in length and have a diameter of 2 to 6 ~; their surface is smooth. The conidia are formed at the apex of the phialides and are oval shapes of 3 to 5 ~ x 5 to 10 ~, and have a smooth surface. These are hyalin at the time of formation, but become blackish green as they mature.

The surface of the conidia is covered with a large amount of viscous material. For this reason, the conidia stick together and form large conidial clusters at the apex of the conidiophores. The viscous material is transparent at the time that it is formed, but then gradually becomes black.

b) Modified Czapek Agar Medium Percent Soluble Starch 1.0 Corn Steep Liquor (dry solids basis) 0.1 NaN03 0.2 K2HP0 4 - 0 . 1 KCl O 05 MgS04 7H20 ~ 0.05 FeS04 7H O 0.001 Agar 2.0 pH to 7.0 with NaOH

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

The colonies are circular and thin, and their surfaces have radiating from their centers a black viscous material which is in the form of oil-like drops having diameters reaching 1 to 3 mm. These arise from the gathering together of clusters of conidia which are enclosed in the viscous material and then form oil-drop-like bodies. The undersides of the colonies show a darker brown color than is seen with the previous Czapek medium, and a small amount of brown pigment is secreted into the medium.

c) Potato-Dex rose Agar Medium The growth of colonies on this medium is a bit slower than on the previously-described Czapek medium, reaching 3 to 4 cm when incubated for 10 days at 30C.
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, developing in a radiating pattern. The surfaces .
of the colonies are black with a slightly green luster, and are rich in hyphae, conidial clusters and so on.

After 14 days of incubation, the surfaces of the old colonies have radiating formations of synnemata standing about l to 3 mm erect. The undersides of these colonies show a blackish-brown color, and a large amount of brown pigment is secreted into the medium.

d) Special Agar Medium Percent Soluble Starch 1.0 Corn Steep Liquor (dry solids basis) 0.2 Cottonseed Oil Dregs 0.1 Yeast Extract 0.1 K2HP04 0.1 MgS04 7H20 0.05 - Agar 2.0 - pH to 7.0 with NaOH

- The colonies on this medium after 10 days of incubation ~t 30C have diameters of 5 to 6 cm, and are round and thin. The vegetative hyphae show good growth and have a black luster. The growth of Gonidia is worse than on the above-mentioned media. The undersides of the colonies are tan in color, and a tan pigment is released into the medium.

e) Davis's Yeast Salt Agar Medium The colonies on this medium after 10 days of incubation at 30C have diameters of 2 to 3 cm znd are more oval than round in shape. The hyphae are tan with ~13~Z

a touch of white and form somewhat thick colonles which are velvety. The undersides of the colonies are tan in color, but absolutely no pigment is secreted into the medium.

10. Physiological Properties of Strain G3~114Q

a) Growth Temperature This strain is capable of growth over a temperature range of 10 to 37C, but its optimum growth temperature is in the vicinity of 30C.

b) Growth pH

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

c) Carbon Source This strain i5 capable of using such carbon sources as dextrose, fructose, galactose, mannose, saccharose, maltose and starch in order to support its growth.

On the basis of the above microbiological findings, strain G30-1140 was identified as Gliobotrys alboviridis after consulting the GENERA OF FUNGI and A MANUAL OF SOIL FUNGI. However, according to the DICTIONARY OF THE FUNGI and G. R. Bisby (Trans. Br.

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Mycol. Soc. 26, 133-43 (1943)), this organism is the same as Stachybotrys subsimpleX, and for this reason strain G30-1140 was identified as Stachybotrys subsimplex.

Strain G30-1140 has conidiophores which stand erect from its vegetative hyphae, branching is almost nonexistent, and they have septa. There are occasions when the basal part is smooth, but the apex is covered with projections. At the apex,a level of phialides form a whorl of 3 to 8 units. Conidia having smooth oblong surfaces divide from these phialides, and they are enclosed in a richly viscous substance. These properties agree well with those described for Stachybotrys subsimplex by G. R. Bisby (Trans. Br. Mycol. Soc. 26, 133-43 (1943)).

This Stachybotrys subsimplex strain G30-1140 is being stored at the Fermentation Research Institute, Agency of Industrial Science & Technology, Chiba City, Japan, as Deposit No. 4377.

Regarding the cultivation of the microorganism to be employed in the present invention, the general knowledge and techniques used in the culture of molds are appllcable.

Namely, as the nutritional source medium, it is possible to employ the media which are used for the culture of ordinary molds. For example, various starches, starch hydrolyzates, corn meal, wheat flour, final molasses, etc., can be employed as carbon sources, while ~17-~ ~ 31~

the nitrogen requlrement can be supplied in the form of peptone, cottonseed oil dregs, meat extract, yeast extract, casein, corn steep liquor, malt extract, soybean dregs, skimmed milk~ inorganic ammonium sa~ts, inorganic nitrates, etc. As the inorganic salts, it is possible to employ calcium chloride, magnesium sulfate, phosphates~ sodium chloride, potassium chloride, and so on. Furthermore, these carbon sources, nitrogen sources and inorganic salts can be used either singly or in appropriate combinations. In addition, when it is desired to promote the growth of the microorganism and bring about an increase in its enzyme production, it is possible to employ trace amounts of metallic salts, vitamins, amino acids, and so forth.

~ The culture conditions usually employed for molds are also applicable to the cultivation of this microorganism. Namely, in liquid culture, if this mlcrobe is cultured for 7 to 14 days at pH 5 to 8 and 20C to 37C together with agitation to provide aeration, the enzyme of the present invention is accumulated in the culture fluid. In addition, if solid materials such as bran are employed, it is possible to carry out solid culture.

Next, an example will be presented of a method whereby the new neutral glucoamylase which is the objective of the present invention can be recovered from the cultured material. In the case of liquid culture, the mycelia are eliminated by any of the publicly-known methods; then the filtrate can be concentrated under reduced pressure, or the enzyme can be salted out with the other proteins by adding inorganic salts such as ammonium sulfate to the filtrate, or the enzyme can be precipitated out and concentrated by the addition of an organic solvent such as acetone or isopropanol.

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

The crude preparations of this new neutral enzyme obtained in this way can then be purified by carrying out the previously-mentioned purification techni~ues.

It is possible to employ this new neutral glucoamylase of the present invention for the saccharl-fication of liquefied starch when producing dextrose from starch. Especially, if the present enzyme is used and the saccharification is carried out at pH 6.o to 6.5, ~here is, as was mentioned earlier, little reverse reaction occurrence, and this results in an increased yield of dextrose being obtainable in comparison with the cases of employing the conventional glucoamylases and carrying out the saccharification under acidic conditions.

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The invention is further illustr~ted by reference to the following examples in which all parts and percentages are by weight unless otherwise noted.

A liquid culture medium containing 5% soluble starch, 2% corn steep liquor, 0.5% cottonseed oil dregs, 0.5% yeast extract~ 0.1% dipotassium phosphate, 0.05%
magnesium sulfate and 0.01% calcium chloride was adjusted to pH 7.0 and 100 ml of this was placed in a 500-ml Erlenmeyer flask. This medium was sterilized at 121C
for 10 minutes, inoculated with Stachybotrys subsimplex strain G30-1140, and incubated at 30C for 7 days on a shaker. After the culture was completed, the mycelia were eliminated from the culture fluid by filtration. The filtrate was found to contain 70 units of glucoamylase activity per milliliter.

This filtrate was next frozen for one night at -20C and then thawed at room temperature. The insoluble matter was removed by centrifugation. Two volumes of cold isopropanol was then added to this solution and it was left standing at 4C for one night so that the enzyme would be precipitated out. The supernatant was removed by decantation and the precipitate was dlssolved in a 0.05 M tris-HCl buffer solution containing 1 mM EDTA
and having a pH of 7.5. This enzyme-containing solution was then dialyzed against the same buf~er at 4~C for one night. DEAE-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 this DEAE-dellulose with the same buffer, the enzyme was eluted from it using a solution of the same buffer containing NaCl at a con~
centration of 0.3 M. Next, two volumes of cold isopropanol was added to the eluate to cause the enzyme to precipitate, and the precipitate was collected by centrifugation. This precipitate was then dissolved in the 0.05 M tris-HCl buffer (pH 7.5j containing 1 mM
EDTA, followed by overnight dialysis against the same buffer solution. The dialyzed enzyme solution was next applied to 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 carried out by linearly increasing the con-centration of NaCl in the same buffer solution up to 0.5 M. The fractions of the eluate which contained the enzyme were then pooled and two volumes of cold iso-propanol was added in order to precipitate the enzyme out of this solution and concentrate it. The concentrated enzyme was next applied to 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 EDTA, and elution was carried out using the same buffer. The eluted fractions 1~311~2 which showed enzyme ac~ ity were then pooled, and two ~olumes of cold isopropanol was added to this to preciPi-tate out the enzyme. This resulted in the recovery of the enzyme in a purified and concentrated form. The specific activity of this purified enzyme was found to be 127 units per milligram of protein.

To a 30% solution of a spray-dried maltodextrin (D.E. about 10) in 0.05 M acetate buffer at pH 6.5 was added the purified glucoamylase of Example 1. The enzyme was added at a dosage of 0.20 units of enzy~e per gram of substrate on a dry solids basis. After the solution had been incubated at 55C for 72 hours, the dextrose content of the filtered hydrolyzate,as determined by high performance liquid chromatography, was 96.5% of the total carbohydrate.

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Starch was converted to a 10.2 D.E. starch hydrolyzate using bacterial alpha-amylase from B.
licheniformis according to the general procedure given in U.S. Patent 3,912,590. The solution was boiled for 5 minutes after adjusting the pH to 2.0 with 2 N HCl to inactivate the residual alpha-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 Exam?le 1 per gram of substrate (dry solids basis). The solution ~3114Z

was incubated at 55C in a stoppered tube. The pH was ad~usted to 6.2 a~ter 5 hours and 48 hours. After the solution had been incubated for 72 hours, the dextrose content of the filtered hydrolyzate,as determined by high performance liquid chromatography, was 97.6p of the total carbohydrate. The final concentration of the solution was 31.2% on a dry solids basis.

When saccharification tests at the same substrate concentration were carried out with commercial glucoamylase from A. niger under its optimum conditions (pH 4.3 at 60C)7 - the corresponding dextrose yield was 96.5%. Similarly, the glucoamylase from R. niveus at pH 5.0 and 55C gave a dextrose yield of 97%. Dextrose yields were about 1~ lower when the saccharification tests were carried out with the commercial glucoamylases under the conditions used for the new enzyme. These results show that the new glucoamylase of this invention gives higher yields of dextrose than do the commercial glucoamylases even when each enzyme is utilized under its optimum reaction conditions.

Claims (7)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A glucoamylase enzyme preparation,prepared by culturing cells of a strain of Stachybotrys subsimplex in a nutrient medium and isolating the glucoamylase enzyme preparation from the culture medium, which comprises a glucoamylase with a molecular weight of about 50,000 as determined by Sephadex G-150 column chromatography.

2. The enzyme preparation of claim 1 wherein the strain of Stachybotrys simplex is Fermentation Research Institute Deposit No. 4377.

3. The glucoamylase enzyme preparation of claim 1 which has an optimum glucoamylase activity in the range of about pH 6.0 to about 6.5 at 60°C and a maximum glucoamylase activity at about 65°C as measured by a 10-minute reaction on a 2% maltodextrin solution at pH 6Ø

4. The glucoamylase enzyme preparation of claim 1 which is capable of converting a 30% by weight solution of a 10 D.E.
starch hydrolyzate to a product containing at least about 96%
dextrose on a dry solids basis when reacted with the starch hydrolyzate at pH 6.0 to 6.5 at 55°C.

5. In a process for producing a syrup of high dextrose content by saccharifying a liquefied starch to dextrose, the improvement which comprises saccharifying the liquefied starch at pH between 6.0 and 6.5 in the presence of the glucoamylase enzyme preparation of claim 1.

6. The process of claim 5 wherein the glucoamylase is obtained from the strain of Stachybotrys subsimplex, Fermentation Research Institute Deposit No. 4377.

7. The process of claim 6 wherein the saccharification is carried out at a temperature of from about 50°C to about 65°C.