JPS6336778A - Heat-resistant glucoamylase and production thereof - Google Patents

Abstract

PURPOSE:To produce a novel glucoamylase having excellent heat-resistance, resistant to acid and especially suitable for the saccharification reaction of starch in the production of glucose, by culturing a strict anaerobe belonging to Clostridium genus. CONSTITUTION:A strict anaerobe belonging to Clostridium genus and capable of producing a novel glucoamylase having excellent heat-resistance and resistant to acid, e.g. Clostridium sp G-0005 (FERM P-8737) is inoculated in a proper medium and cultured at about 60 deg.C for 1-3 days under anaerobic condition. The objective novel heat-resistant glucoamylase is separated from the cultured liquid and cultured cells.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention relates to a novel glucoamylase and a method for producing the same, and in particular, a heat-resistant/acid-resistant glucoamylase suitable for the saccharification reaction of starch in glucose production and its production. Regarding the law.

[Prior art]

Thermostable enzymes are more stable against heating and pH changes than enzymes used at room temperature, and are extremely useful in enzyme utilization industries.

Glucoamylase currently used industrially is produced by Rhizopus (Rh 1zopus) and Aspergillus (
They are produced from the genus Aspergillus 1us), and both are room-temperature enzymes, and their enzyme activity rapidly deactivates when the temperature exceeds 50°C (Enzyme Utilization Handbook, p. 62, Nishinshokan, 1982). Regarding glucoamylase with excellent heat resistance, V, Basaveswara et al.
Hem, J., 193.379.1981) and cadocosin (Japanese Patent Application Laid-open No. 60-54680), but in order to exhibit thermal stability, neutral conditions of p1) 6 are required. is necessary. By the way, in the production of glucose using starch as raw material, starch slurry with a concentration of 10 to 40% is
- liquefaction step of liquefying with amylase and saccharification step of saccharifying this liquefied starch solution with glucoamylase.
This is done through a process. In the liquefaction process, the pH is 5 or less, often 4 or less, due to impurity organic acids contained in the raw starch. For this reason, the present inventors previously developed a new α-
Special Application No. 236917/1983 for the development of amylase),
This made it possible to liquefy starch slurry in an acidic state. However, glucoamylase used in the saccharification reaction that follows the liquefaction process has a pH of 4 to
5. Thermostable enzymes that can act under acidic conditions have not yet been developed. The preferred pH for action of the above-mentioned thermostable glucoamylase such as cadocosin is around 6, and therefore it is necessary to neutralize it by adding an alkali to the liquefied starch solution. As a result, when obtaining final products such as glucose and high fructose sugar, it is necessary to remove the neutralizing agent after the reaction, which significantly increases the load on the desalting process using an ion exchange resin.

[Problem that the invention seeks to solve]

The purpose of the present invention is to solve the problems of the prior art described above, and to provide a glucoamylase that can be used under conditions of pH 5 or less and 60 to 75°C in the production of grapes, etc. using starch as a raw material, that is, obligate anaerobic bacteria. The object of the present invention is to provide a novel glucoamylase, which originates from . and has not only excellent heat resistance but also acid resistance, and a method for producing the same.

[Technical means to solve problems]

The present inventors conducted a search for enzymes and enzyme-producing microorganisms with the aim of obtaining a novel glucoamylase that has excellent heat resistance and acid resistance.

As a result, Clostridium sp. G-0005 (clostridium sp.
ridium sp G-0005)
No. 737) discovered that a novel thermostable glucoamylase with enzyme properties completely different from conventional glucoamylases was produced, leading to the present invention. The bacterium of the genus Clostridium that produces the glucoamylase of the present invention has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology (Accession number: Fiber Science and Technology Research Institute No. 8737 (14RM P-8737)). First, the details of Honda's mycological properties will be explained.

In the following description, % is by weight unless otherwise specified.

A. Morphological properties (1) Morphology of vegetative cells When cultured on an agar plate in the following dextrin-peptone medium in an anaerobic atmosphere at 60°C for 2 days, the vegetative cells are 0.3-0.5 x 2- It is a straight bacillus with a size of 4 μm. The above vegetative cells exist singly or in chains. The same applies to liquid culture. Moreover, no cell pleomorphism is observed.

Dextrin-peptone medium composition Dextrin 1.5% peptone
0.5% yeast extract
0.5% Kll□PO40,7% Na1l! P0. 0.2%Mg
5O, 7H, 00,001% Agar 2.0% Sodium thioglycolate 0.1% Tap water pH 6.0 (2) Mobility No motility observed (3) Spores present Starch-peptone medium Spore formation is observed in agar plate culture. Spores are formed at the inner end of vegetative cells and are spherical with a diameter of about 0.5 μm.

(4) Durham staining negative B, production status in each medium (1) Colony morphology Colonies in agar plate culture of starch-peptone medium are as follows:
It has a flat circular shape with a slightly raised center and irregular edges. It does not produce any pigment and is milky white and translucent.

(2) Meat juice agar plate culture No growth observed Meat juice agar medium Composition Meat extract 1.0% peptone
1.0% salt 0.2% sodium thioglycolate 0.1% agar
2.0% Distilled water pH 6.0 (3) No growth observed in broth agar slant culture (4) Broth agar puncture culture Slight growth is observed along the puncture line. No pigment production is observed.

(5) Meat juice liquid culture Composition of meat juice medium where no growth is observed Meat extract 1.0% peptone
1.0% salt 0.2% sodium thioglycolate 0.01% distilled water pH 6.0 (6) No growth of meat juice/gelatin culture was observed. No liquefaction of gelatin was observed.

Composition of meat juice gelatin medium Meat extract 1.0% peptone
1.0% salt 0.2% gelatin 15.0% sodium thioglycolate 0.1% distilled water pH 6.0 (7) Lidomus milk culture produces acid, turns red, and solidifies (coagulates. Gas is generated. Is recognized.

C0 Physiological properties (1) Growth temperature range: Grows at 49-64°C. No growth was observed at 46°C and 69°C. It grows well around 57-61°C.

(2) Growth pH range Grows at pH 4.8 to 7.5. Grows well around pH 6.0.

(3) Anaerobic attitude toward oxygen (4) O-F test (Hugh La1fson method)
negative. It grows with gas production both in air atmosphere and under anaerobic conditions with liquid paraffin layer, and turns yellow due to acid production. When cultured in an air atmosphere, growth occurred from about 1 cs below the gas phase boundary to the bottom.

Medium composition Peptone 0.2% glucose
1.0% salt 0.5% 2HP0. 0.03% bromcresol purple 0.002% agar
0.3% Distilled water pH 6.0 (5) Nitrate reduction negative (6) VP test negative (7) MR test positive. Changes to red.

(8) Indole production It cannot be measured because it does not grow in peptone water.

(9) Negative for the production of hydrogen sulfide (when using Kligrer's medium) (10) Positive for starch hydrolysis (1)) Positive for the use of citric acid (when using Simons medium) (12) Use of ammonium salts When grown in peptone water It cannot be measured.

(13) Negative for extracellular production of pigment (14) Negative for urease (15) Negative for oxidase (16) Negative for catalase (17) Observation results of assimilation of assimilated sugars of ttJM and presence or absence of gas generation using Durham tube are shown in Table 1. In the table, when assimilation and gas production were observed, it was indicated by a + symbol, and when it was not observed, it was indicated by a - symbol.

(Margins below this page) Table 1 Composition of liquid medium for glycosylation test Carbon source (sugar) 1.0% Peptone 1.0% NaC 10,2
% Sodium thioglycolate 0.1% Distilled water pH 6.0 (18) Growth on inorganic salt medium No growth was observed.

Based on these results, Burgee's bacterial classification manual (Be
rgey's Manual of Determin
Active Bacteri-ology 8th E
It was identified as a bacterium belonging to the genus Clostridium.

Next, the enzymatic properties of the thermostable glycoamylase of the present invention will be described.

Note that glucoamylase activity was measured as follows.

Soluble starch (manufactured by Wako Pure Chemical Industries, Ltd., for biochemical use) was used as the substrate for measuring glucoamylase activity. First, 0.5 d of 5% soluble starch solution, 0.5 d of 0.1 M acetic acid-sodium acetate buffer with pH 4.5, and 1 ml of pure water.

0.5 - of the enzyme solution was mixed and the enzyme reaction was carried out at 60°C for 30 minutes. Next, the glucose in the reaction solution was measured using a glucose analyzer (Yellow Springs Instrument Company, USA).
Measured by the glucose oxidase method manufactured by Glucose Instrument Company. ).

One unit of glucoamylase activity is 1 unit of glucoamylase activity under the above measurement conditions.
It was defined as the ability to produce 1 μmol of glucose per minute.

(1) Preparation method Since it will be explained in detail in Examples, only a brief explanation will be provided here.

Clostridia bacterium G-0005 according to the present invention is inoculated into a liquid medium containing dextrin/peptone and yeast extract, and cultured at 60° C. for 1 to 3 days under anaerobic conditions. A so-called culture filtrate is obtained by removing bacterial cells and other insoluble substances from the culture solution by centrifugation or the like. Then,
The glucoamylase of the present invention is concentrated from the culture filtrate using appropriately known methods such as molecular sieve membrane filtration, salting out ion exchange chromatography, gel filtration chromatography, etc.
Remove other impurities.

In addition, the carbon source of the liquid medium in liquid culture is not limited to the above-mentioned dextrin, and soluble starch, potato starch, corn starch, sweet potato starch, waste molasses, etc. may also be used. Further, other nutritional components are not limited to those mentioned above, and cornstarch liquor, various amino acids, vitamins, various salts, etc. may be used alone or in combination.

(2) Action and substrate specificity This enzyme is a glucoamylase that hydrolyzes starches such as potato, corn, and sweet potato, as well as soluble starch obtained by hydrolyzing these, dextrin, maltose, etc., into glucose. For maltose substrates, the rate of glucose production is approximately 2 times that of soluble starch substrates.

(3) Optimal pH FIG. 1 shows the action pH curve of this enzyme at 60°C.

The optimal pH range of this enzyme at 60°C is 4 to 5, and the preferred pH is (pH having 80% of the activity at the optimal pH).
H range) ranges from 3.8 to 5.7.

In addition, as a pH buffer during the reaction, potassium chloride-
Hydrochloric acid (pH 2.0), glycine-hydrochloric acid (pH 2.5~
3.5), β:β-dimethylglutarate-tris(hydroxymethylamino)methane-2-amino-2-methyl-1:3-propanediol (hereinafter abbreviated as rGTAJ)
A 0.05M buffer (pH 3,5-9) was used.

(4) pH stability The glucoamylase of the present invention has a pH of 2, 3, 4°4.5,
Under each pH of 5.6 and 7.9 (0.05M potassium chloride-
Hydrochloric acid, 0.05M glycine-hydrochloric acid and 0.05MG
The cells were incubated at 70° C. for 30 minutes. After that, the reaction solution was diluted to pH 4.5.
The remaining activity was measured using soluble starch as a substrate. The results are shown in Figure 2. The glucoamylase of the present invention completely retained its activity within the pH range of 4.5 to 5.0. Therefore, this glucoamylase was found to be an enzyme with excellent stability in an acidic region.

(5) Optimal temperature The activity of the glucoamylase of the present invention was measured at a temperature of 40.50.60.65.70.75°C under the condition of pH 4.5, and the results shown in Figure 3 were obtained. . From this result,
The optimum temperature is around 70°C. The preferred temperature (temperature range having 80% of the activity at the optimum temperature) is 53-73°C
It is.

(6) Effect of metal salts on glucoamylase activity Table 2 shows the effects of metal salts on the activity of glucoamylase of the present invention. In measuring glucoamylase activity, various metal salts were added at 5mM8. The activity was expressed in % relative to that without addition of metal salts. In addition, the case of addition of ammonium salt and EDTA is also added to Table 2. magnesium ions, calcium ions,
It is recognized that potassium ions have a glucoamylase activating effect. Nickel ions and iron ions have an inhibitory effect.

(Leaving space below next summer) Table 2 Activity measurement conditions pH 5.0 (0.1M citric acid-secondary sodium citrate buffer) Activity measurement temperature: 60"C (7) Thermal stability Using the glucoamylase of the present invention as a substrate Without additives, pH 4.5
Heat treatment was performed at 60 to 80°C at regular intervals (2
0.40 seconds, 1. 2. 4.10.20.40 minutes, 1°2.4.6,8.16 hours, 1.2.4.7.1
A portion of the treated solution was collected on 0°15 and 20 days), and the glucoamylase activity in the solution was measured at 5Q'C and pH 4.5.

Based on this, the activity half-life at each temperature was determined, and the results are shown in FIG. The half-lives of activity in the absence of substrate at 70°C and 75°C are 6 hours and 1 minute, respectively. This glucoamylase has a higher degree of thermostability than conventionally known glucoamylases. On the other hand, pH4
．． 0, 4.3, 4.5, 5,0.

The half-lives of activity at 70'C without the addition of substrate at each pH of 6.0 were determined and shown in Table 3. From this result, the present glucoamylase is derived from Clostridium thermoamylolidicum (Clostridium thermoamylolidicum).
yloly-ticum), Thermomyces lanuginosus (Thermo-myces 1anugino)
susL and Taracomyces 0 du Ponti (Talar
It exhibits superior heat resistance, especially in the acidic range of pH 4 to 5, than glucoamylase produced by S. omyces duponti.

(Margins below this page) (8) Effect of metal salts on heat resistance Table 4 shows the effects of metal salts on the heat resistance of the glucoamylase of the present invention. Aqueous solution of glucoamylase (0,0
Dissolved in 5M acetic acid-sodium acetate buffer, pH 4.5)
Various metal salts were added to the solution at a concentration of 5mM, and 70
After heat treatment at ℃ for 1 hour, pH 4.5, 60
Glucoamylase activity was measured at °C. Then, the activity after heat treatment compared to before heat treatment, that is, the residual activity, was calculated as %.
It was displayed in

From Table 4, it is recognized that nickel ions, manganese ions, and magnesium ions have a protective effect. No protective effect was observed for cobalt ions and calcium ions. Zinc ions significantly reduce heat resistance.

(Margins below this page) Table 4 (9) Molecular weight The molecular weight of the glucoamylase of the present invention was determined by gel filtration chromatography (Sephadex G-1 manufactured by Pharmacia, Sweden).
It is determined to be 3.8 x 10' from the elution pattern (using 00).

[Function]' As is clear from the above, the new thermostable glucoamylase of the present invention is superior to conventional thermostable enzymes produced by anaerobic bacteria, especially in terms of heat resistance in the acidic region of pH 4 to 5. Significantly different.

By the way, in order to produce glucose and high fructose sugar, first,
The raw material starch is liquefied with α-amylase, and then saccharified with glucoamylase. During liquefaction, the raw material starch is charged at a high concentration of 20 to 40%, so the pH of the liquid becomes acidic. For this reason, when using conventional thermostable α-amylase and thermostable glucoamylase, the action pH range is in the neutral range, so they must be neutralized with an alkali.

In contrast, a new heat-resistant and acid-resistant α-amylase previously discovered by the present inventors (Japanese Patent Application No. 59-236917
By using the novel glucoamylase of the present invention, it becomes possible to carry out liquefaction and saccharification in an acidic state without neutralization during liquefaction, and as a result, it becomes possible to carry out liquefaction and saccharification in an acidic state without performing neutralization during liquefaction. The load can be significantly reduced.

〔Example〕

Hereinafter, examples of the present invention will be shown and explained in more detail.

Example 1 Dextrin 1.5%, polypeptone 0.5%, yeast extract 0.5%, monopotassium phosphate 0.7%, dibasic sodium phosphate 0.2%, magnesium sulfate heptahydrate 0
．． 001%, sodium thioglycolate 0.1%, and tap water (pH 6,0), 32 kg was dispensed into 10 culture tanks each having an internal volume of 51, each weighing 3.15 kg.
It was sterilized at 120°C for 15 minutes. To each culture tank was added 0.35 kg of a cell suspension of Clostridium bacteria G-0005 that had been anaerobically cultured using the above medium.

Next, a water seal trap was attached to the gas outlet, and the gas phase in the culture tank was sufficiently replaced with argon gas (oxygen concentration i ppm or less), followed by culturing under anaerobic conditions. The pH of the culture solution was automatically adjusted to 6.0, and the temperature of the culture solution was automatically adjusted to 60°C. 21
After culturing for an hour, the culture solutions were combined and centrifuged at 6000 rpm to remove the bacterial cells. Glass filter paper (Toyo Kagaku Sangyo type, GA-100 and GC-50) was used for the collected supernatant liquid.
Membrane filter (Toyo Kagaku Sangyo type, pore size 1
Microbial cells and other solid matter were removed by pressure filtration using microbial filters (μm and 0.45 μm) to obtain a culture filtrate. After collecting IO- from this culture filtrate and dialyzing it against pure water for 24 hours, glucoamylase activity was measured.
There were 0.05 units of glucoamylase per g.

Next, 29 ksr of the culture filtrate was filtered under pressure using a hollow fiber molecular sieve membrane (fraction molecule ff1lo, 000. HIPIO-20, manufactured by Amicon Co., USA) and concentrated to 2 kg. The above concentrated solution was subjected to salting out using ammonium sulfate, and a solid substance that did not precipitate at 20% saturation (ratio to the saturated ammonium sulfate concentration, expressed as %) but precipitated at 55% saturation was determined in step 14. It was centrifuged and collected at OOOrpm. The solids were then washed and collected with a 55% saturated ammonium sulfate solution. Next, the solid matter was dissolved in 0.05M) Lis-HCl buffer (pH 7.5) to give a total weight of 240 g. Next, dialysis for 24 hours was repeated twice using the above buffer 10β. Thereafter, solid matter in the dialyzed solution was removed by filtration using glass filter paper (GC-50, manufactured by Toyo Roshi). The clarified dialysate weighed 310 g.

Next, the above dialysate was mixed with diethylaminoethylated cross-linked agarose gel (manufactured by Pharmacia, DEAR Sepharose C).
Purification was performed by ion exchange chromatography (column size φ44 x 500 mm) using L-68). 0.05
The above dialysate was charged onto a gel column equilibrated with M Tris-HCl buffer and washed. The sodium chloride concentration in the buffer was then developed in an increasing linear gradient. The elution pattern of glucoamylase is shown in FIG. A peak with glucoamylase activity was observed at the elution position at a sodium chloride concentration of 0.25M. 360 g was collected as a glucoamylase fraction, and then a molecular sieve membrane (molecular weight cutoff 10,000, manufactured by Amicon, USA,
PM-10), add 10 kg of pure water, filter under pressure, desalt, and concentrate to obtain 160 g of crudely purified glucoamylase solution.
I got it. The glucoamylase activity in this solution was 3.3 units/g.

Next, the crudely purified glucoamylase was purified by gel filtration. First, add 100g of the above gelcomylase solution to 40g
The product was lyophilized under reduced pressure of Torr, dissolved in 4 d of 0.05 M citric acid/second citrate buffer (pH 4.5), and the solid matter was removed by centrifugation at 400 Orpm.

Next, cross-linked dextran gel (
The above glucoamylase solution 1- was charged into a chromatography column (φ15 x 900 mm) packed with Sephadex G-100 (manufactured by Pharmacia) and developed with the same buffer. The results are shown in FIG. Eluent ff165mZ
A peak of glucoamylase activity was observed at the position.

The above gel filtration was also performed on the remaining crudely purified glucoamylase to obtain 40 g of purified glucoamylase fraction. The glucoamylase activity of this fraction was 12 units/g.

Through the above purification procedure, the specific activity based on the culture filtrate was 171.
improved twice. In addition, the activity recovery rate based on culture filtrate was 23
%. Table 5 shows the specific activity, yield, and activity recovery rate of the preparation in each step based on the culture filtrate.

(Margins below this page) Example 2 In Example 1, 400 g of solid matter containing bacterial cells was collected from the culture solution by centrifugation. Then, physiological saline 60
Add 0g to make a slurry, and make 1000g of bacterial cell slurry.
I got it. Next, 40 g was taken from the bacterial cell slurry and placed in a pressure cell for a French press (manufactured by Otake Seisakusho).

Next, a pressure of 1,600 kg/cnl was applied using a hydraulic press, and the microbial cells were crushed by ejecting into the atmosphere from the narrow opening at a speed of 5 to 7 minutes.

The above operation was performed for the remaining bacterial cell slurry to obtain 1000 g of crushed bacterial cell slurry.

Next, solid matter was removed from the bacterial cell slurry by centrifugation at 16.00 rpm, and glass filter paper (Toyo Roshi Layer, GA-100, GC-50) and membrane filter (Toyo Roshi Layer, pore size 1, 0.45 μm) were filtered. 800 d of crushed bacterial cell supernatant was obtained.

Next, the supernatant of the disrupted bacterial cells was heat-treated at 70°C for 5 minutes to denature proteins that tend to be thermally denatured. After cooling to below 5°C, solid matter was removed by centrifugation at 8000 rpm. The heat-treated supernatant obtained had a weight of 760 d.

Next, purification by ammonium sulfate precipitation, dialysis, ion exchange chromatography, and gel filtration was performed using the method described in Example 1. The specific activity, yield, and
The activity recovery rate is shown in Table 6.

(blank below unfaithfulness) Example 3 Corn starch 2.3%, polypeptone 0.2%.

Yeast extract 0.2%, ammonium sulfate 0.2%, monopotassium phosphate 0.2%, magnesium sulfate heptahydrate 0
20kg of a liquid medium (pH 6,4) containing 0.002% cysteine, 0.1% cysteine, and tap water was placed in a culture tank with an internal volume of 50β, steam was blown into the jacket part provided inside the tank and on the outer wall of the culture tank, and 120 Sterilization was carried out at ℃ for 20 minutes.

Next, the inside of the culture tank was cooled to 60°C, and sterile water was added to make the culture medium volume 32kir. Next, 3.5 kg of Clostridium G-0005 bacterial suspension prepared by the culture medium and method shown in Example 1 was added. Next, a water seal trap was attached to the gas outlet, and after replacing the gas phase in the culture tank with nitrogen gas, the temperature was 60°C and pH 6 under anaerobic conditions.
, 5. After culturing for 40 hours, solid matter was immediately removed using a continuous centrifuge to obtain a culture supernatant of 33.5 kg.
I got g. The main culture supernatant contained 0.09 units of glucoamylase activity, 7 g. Next, 21 of the above culture supernatant was placed in a low evaporator flask, and 6
Vacuum distillation was performed using a rotary evaporator while immersed in a 0°C water bath. In order to suppress foaming at the start of distillation, pressure reduction was started after n-octyl alcohol l- was added. Depending on the amount of decrease in the culture supernatant in the flask, new culture supernatant was supplied from the outside into the flask, and vacuum distillation was continued for m. Note that n-octyl alcohol was added at any time when foaming in the flask became significant. 2.3 p of a concentrated solution was obtained by distillation under reduced pressure.

Next, the concentrate was heat treated at 70°C for 5 minutes, and immediately cooled to 50°C or lower. Then 700
Solid matter precipitated by heat treatment was removed by centrifugation at 0 rpm to obtain heat-treated supernatant 2.1).

Next, the heat-treated supernatant liquid was subjected to salting out using ammonium sulfate, and both fractions with a saturation degree of 20% to 55% were recovered. After washing this with ammonium sulfate solution having a saturation degree of 55%, the solid matter was dissolved in distilled water to obtain 520 g of a crude glucoamylase solution.

Next, crude glucoamylase? Yong? Cornstarch 2 at night
After adding 00 g and stirring and mixing thoroughly, corn starch was separated by centrifugation and a supernatant liquid was collected. Furthermore, the corn starch was washed with 200 g of distilled water, the washing liquid was collected, and together with the above supernatant liquid, 490 g of a starch adsorption treatment liquid was obtained. Next, glass filter paper (manufactured by Toyo Roshi Co., Ltd., GA-
100, GC-50) to obtain 480 g of a crude glucoamylase solution. The glucoamylase activity of this solution was 5.3 units/g, and the specific activity was 59 times higher than that of the culture supernatant, and the activity recovery rate was 84%.

〔Effect of the invention〕

The novel heat-resistant/acid-resistant glucoamylase of the present invention has excellent heat resistance, especially in the acidic range of pH 4 to 5, so it can be used to produce glucose from starch without neutralizing the starch solution. Saccharification is possible under acidic conditions. In contrast, when using conventionally known thermostable glucoamylases, an alkali must be added to neutralize the starch solution. Therefore, by using the glucoamylase of the present invention, the step of neutralizing the starch solution becomes unnecessary, and the load on the desalting step after the reaction can be significantly reduced.

[Brief explanation of drawings]

Figure 1 shows the effect of pH on the activity of glucoamylase of the present invention.
Figure 2 is a characteristic diagram showing the influence of pH on the thermostability of this glucoamylase, Figure 3 is a characteristic diagram showing the influence of temperature on the activity of this glucoamylase, Figure 4 is a characteristic diagram showing the heat resistance of this glucoamylase,
Figure 5 is a glucoamylase activity elution pattern diagram of this glucoamylase in an ion exchange liquid chromatograph using diethylaminoethyl cross-linked agarose gel.
The figure is a diagram showing the glucoamylase activity elution pattern of the present glucoamylase in gel filtration using a cross-linked dextran gel. Patent applicant Hitachi, Ltd. Representative Patent attorney Yusuke HirakipH Figure 2H Figure 4

Claims (1)

[Claims] 1. A thermostable glucoamylase exhibiting the following physicochemical properties. (1) Optimum pH for action: 4-5 (2) Optimum temperature for action: 70°C (3) Stable pH: 4.5-5.0 (4) Half-life of enzyme activity at pH 4.5 and temperature 70°C 6 hours or more (5) Enzyme activity half-life at pH 4.0 and temperature 70°C is 5.5 hours (6) Molecular weight: 3.8 x 10^4 2, glucoamylase belonging to the genus Clostridium and having the following physicochemical properties 1. A method for producing thermostable glucoamylase, which comprises culturing a producing bacterium, accumulating the glucoamylase in the bacterium and culture medium, and recovering the glucoamylase from the bacterium and/or the medium. (1) Optimum pH for action: 4-5 (2) Optimum temperature for action: 70°C (3) Stable pH: 4.5-5.0 (4) Half-life of enzyme activity at pH 4.5 and temperature 70°C 6 hours or more (5) Enzyme activity half-life at pH 4.0 and temperature 70°C is 5.5 hours (6) Molecular weight: 3.8 x 10^4 3. Glucoamylase-producing bacteria belonging to the genus Clostridia 3. The method for producing a thermostable glucoamylase according to claim 2, wherein the glucoamylase is G-0005.