JPH0425794B2 - - Google Patents

Description

[Detailed description of the invention]

[Field of Industrial Application] The present invention relates to a novel anaerobic bacterium used for the production of glucoamylase, and more specifically, the present invention relates to an anaerobic bacterium used for the production of a novel glucoamylase, and more specifically, the present invention relates to an anaerobic bacterium that is excellent even under acidic conditions suitable for the saccharification reaction of starch in the production of glucose, etc. The present invention relates to Clostridium sp., which produces thermostable/acid-stable glucoamylase. [Prior art] Enzymes have high substrate specificity and can catalyze reactions even at room temperature and pressure;
Extremely unstable to change. By immobilizing bacteria and enzymes and incorporating them into a bioreactor, it has become possible to produce isomerized sugar and L-amino acids. When operating these reactors, it is desirable to operate them at a temperature of 60°C or higher, which is higher than room temperature, in order to prevent the propagation of bacteria and increase reaction speed. For this reason, efforts have been made to develop so-called thermostable enzymes, which are stable against heat and pH changes, in place of conventional room-temperature enzymes. Glucoamylase, which is currently used industrially, is a room-temperature enzyme whose enzymatic activity rapidly deactivates when the temperature exceeds 50°C (Enzyme Utilization Handbook, p. 62).
It is mainly produced from the genus Rhizopus and Aspergillus. The preferred pH for the action of these glucoamylases is 4 to 5. Regarding glucoamylase with excellent heat resistance,
V. Basaveswara et al. (Biochem.J., 193, 379,
1981) and katokosin etc. (JP-A-60-54680)
There are examples of these, but all require neutral conditions of about PH6 in order to exhibit thermal stability. By the way, the production of glucose using starch as a raw material is carried out through two steps: a liquefaction process in which a starch slurry with a concentration of 10 to 40% is liquefied using α-amylase, and a saccharification process in which this liquefied starch solution is saccharified using glucoamylase. . In the liquefaction process, due to the impurity organic acids contained in the raw starch,
PH is below 5, often below 4. For this reason, the present inventors previously developed a new α-amylase that has excellent heat resistance even at temperatures around 4 (Japanese Unexamined Patent Publication No. 59-236917), and developed a new α-amylase that can be used in acidic conditions without neutralizing starch slurry with alkali. This made it possible to liquefy it while it was still in use. However, regarding glucoamylase used in the saccharification step that follows the liquefaction step, a thermostable enzyme that can perform saccharification under acidic conditions of pH 4 to 5 has not yet been developed. Since the preferable pH for action of heat-stable glucoamylase such as catocosin mentioned above is around 6, it is necessary to neutralize the liquefied starch solution by adding an alkali to carry out the saccharification process. As a result, when obtaining final starch products such as glucose and high fructose sugar, it is necessary to remove the neutralizing agent after the reaction, which significantly increases the negative impact on the desalting process using ion exchange resin. ing. [Problems to be Solved by the Invention] The purpose of the present invention is to solve the above-mentioned problems of the prior art, and to produce grapes etc. using starch as a raw material, under acidic conditions of PH4 to 5 and at high temperatures of 60 to 70%. Glucoamylase capable of acting under
An object of the present invention is to provide a microorganism that produces a novel glucoamylase with excellent acid resistance. [Technical means for solving the problem] The present inventors searched for a microorganism that produces a novel glucoamylase that has excellent heat resistance and acid resistance. As a result, Clostridium sp G-0005 (Clostridium sp G-0005), an obligate anaerobic bacterium belonging to the Clostridium family [Feikoken Jyoyori No. 1455 (Feikoken Bichiyori No. 8737)] discovered that they can produce a novel glucoamylase that differs from conventional glucoamylases in terms of enzymatic properties, particularly heat resistance in an acidic region, leading to the present invention. The bacteria of the genus Clostridia of the present invention are isolated from a high-temperature methane fermentation slurry of concentrated organic waste liquid. Isolation of this bacterium was performed as follows. First, the methane fermentation slurry was subjected to low-speed centrifugation (1000 rpm, 5 minutes) to remove large particles by sedimentation, and then diluted with sterile physiological saline. This bacterial solution was applied to an agar plate using starch granules as a carbon source under a nitrogen atmosphere, and colonies that grew anaerobically at 60°C without dissolving the starch granules were isolated.
Furthermore, vegetative cells were isolated from the diluted solution of the colony using a micromanipulator. Separation using an agar plate and separation using a micromanipulator were repeated several times to obtain the bacteria of the present invention. The Clostridium bacterium of the present invention has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology. [Deposit number: Microtech Research Institute No. 1455 (Feiko Research Institute No. 8737)]. first,
The details of the mycological properties of this bacterium are explained below. 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, vegetative cells have a size of 0.3 to 0.5 x 2 to 4 μm. It is a straight bacillus. The above vegetative cells exist singly or in chains. The same applies to liquid culture. Moreover, no cell pleomorphism is observed. A microscopic photograph of the biological form of this bacterium is shown in FIG. Dextrin-peptone medium composition Dextrin 1.5% Peptone 0.5% Yeast extract 0.5% KH ₂ PO ₄ 0.7% Na ₂ HPO ₄ 0.2% MgSO ₄・7H ₂ O 0.001% Agar 2.0% Sodium thioglycolate 0.1% Tap water PH6. 0 (2) Presence or absence of motility No motility observed (3) Presence or absence of spores Formation of spores is observed in agar plate culture of starch-peptone medium. Spores are formed at the inner end of vegetative cells and are spherical with a diameter of about 0.5 μm. (4) Gram staining Negative B Growth status in each medium (1) Colony morphology Colonies cultured on agar plate in starch-peptone medium are flat circular 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 PH6.0 (3) Meat juice agar slant culture Growth (4) Meat juice agar puncture culture Slight growth is observed along the puncture line. No pigment production is observed. (5) Meat juice liquid culture No growth observed Meat extract 1.0% Peptone 1.0% Salt 0.2% Sodium thioglycolate 0.01% Agar 2.0% Distilled water PH6.0 (6) Meat juice/gelatin culture Growth is unacceptable. 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 PH6.0 (7) Litmus milk culture Produces acid, turns red, and solidifies. Gas generation is observed. C Physiological substances (1) Growth temperature range: Grows at 49-64℃. No growth was observed at 46℃ and 69℃. Grows well around 57-61℃. (2) Growth PH range: Grows at PH4.8 to 7.5. Grows well around PH6.0. (3) Attitude towards oxygen Obligate anaerobic (4) O-F test (Hugh Laifson method) Negative. It grows with gas production both in the air atmosphere and under anaerobic conditions using a layer of liquid paraffin, and turns yellow due to the production of acid. When cultured in an air atmosphere, growth occurred from about 1 cm below the gas phase boundary to the bottom. Medium composition Peptone 0.2% Glucose 1.0% Salt 0.5% K ₂ HPO ₄ 0.03% Bromcresol purple 0.002% Agar 0.3% Distilled water PH6.0 (5) Nitrate reduction Negative (6) VP test Negative (7) MR test Positive. Changes to red. (8) Indole production Cannot be measured because it does not grow in peptone water. (9) Hydrogen sulfide production Negative (when using Kligrer's medium) (10) Starch hydrolysis Positive (11) Use of citric acid Positive (when using Simons medium) (12) Use of ammonium salts Does not grow in peptone water , cannot be measured. (13) Extracellular production of pigment negative (14) Urease negative (15) Oxidase negative (16) Catalase negative (17) Sugar assimilation Observation results of sugar assimilation 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.

[Table] Composition of liquid medium for sugar assimilation test Carbon source (sugar) 1.0% Peptone 1.0% NaCl 0.2% Sodium thioglycolate 0.1% Distilled water PH6.0 (18) Growth on mineral salt medium No growth observed . Based on these results, Bergey's Manual of Determinative
It was identified as a bacterium belonging to the genus Clostridium based on the 8th Edition (Bocteriology 8th Edition). Next, the enzymatic properties of the thermostable glycoamylase produced using the bacteria 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,
0.5ml of 5% soluble starch solution, 0.1M acetic acid at PH4.5
0.5 ml of sodium acetate buffer, 1 ml of pure solution, and 0.5 ml of enzyme solution were mixed, and an enzyme reaction was performed at 60° C. for 30 minutes. Next, the amount of glucose in the reaction solution was measured using a glucose analyzer (Yellow Springs Instrument Company, USA).
Measured by the glucose oxidase method manufactured by Instrument Company. ).
One unit of glucoamylase activity was defined as the ability to produce 1 μmol of glucose per minute under the above measurement conditions. (1) Preparation method Dextrin 1.5%, polypeptone 0.5%, yeast extract 0.5%, potassium phosphate 0.7%, dibasic sodium phosphate 0.2%, magnesium sulfate heptahydrate 0.001%, sodium thioglycolate 0.1% and a liquid medium containing tap water (PH
6.0) 3.15Kg from 32Kg to 10 culture tanks with internal volume 5
The solution was divided into portions and sterilized at 120°C for 15 minutes. To each culture tank was added 0.35 kg of a cell suspension of Clostridium G-0005 bacteria of the present invention, which 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 1 ppm or less), followed by culturing under anaerobic conditions. The pH of the culture solution is
6.0, the temperature of the culture medium was automatically adjusted to 60 °C, respectively. After culturing for 21 hours, combine the culture solution and
The cells were removed by centrifugation at 6000 rpm. The collected supernatant liquid was filtered using glass filter paper (manufactured by Toyo Kagaku Sangyo, GA-100 and GC-50) and membrane filter (manufactured by Toyo Kagaku Sangyo, pore size 1 μm and
0.45 μm) to remove bacterial cells and other solids to obtain a culture filtrate. When 10 ml of this culture filtrate was collected and dialyzed against pure water for 24 hours, glucoamylase activity was measured, it was found that 0.05 units of glucoamylase were present per 1 g of culture filtrate. Next, the above culture filtrate 29
Kg is a hollow fiber type molecular sieve membrane (molecular weight cut off 10000, manufactured by Amicon Co., USA)
The mixture was filtered under pressure using H1P10-20) and concentrated to 2 kg.
The above concentrated solution was subjected to salting out using ammonium sulfate,
A solid substance that did not precipitate at a saturation level (ratio to ammonium sulfate saturation concentration, expressed as %) of 20% but precipitated at a saturation level of 55% was centrifuged at 14,000 rpm and collected.
The solids were then washed and collected with a 55% saturated ammonium sulfate solution. Next, the solid substance was dissolved in 0.05M Tris-HCl buffer (PH7.5) to give a total weight of 240 g. Next, dialysis was repeated twice for 24 hours using the above buffer solution 10. After that, the solids in the dialyzed solution were filtered using glass filter paper (Toyo Roshi Co., Ltd., GC-50).
was removed by filtration using The clarified dialysate
It was 310g. Next, the above dialysate was added to a diethylaminoethylated cross-linked agarose gel (manufactured by Pharmacia, DEAE).
It was purified by ion exchange chromatography (column size φ44 x 500 mm) using Sepharose CL-6B). The dialysate was charged to a gel column equilibrated with 0.05M 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 of glucoamylase fraction was recovered.
Next, molecular sieve membrane (molecular weight cutoff)
10000, manufactured by Amicon, USA, PM-10),
10 kg of pure water was added, filtered under pressure, desalted, and concentrated to obtain 160 g of a crudely purified glucoamylase solution. Glucoamylase activity in this solution is 3.3 units/g
It was hot. Next, the crudely purified glucoamylase was purified by gel filtration. First, 100 g of the above glucomylase solution was freeze-dried under reduced pressure of 0.04 Torr.
This was dissolved in 4 ml of 0.05M citric acid/second citrate buffer (PH4.5), and solid matter was removed by centrifugation at 4000 rpm. Next, cross-linked dextran gel (manufactured by Pharmacia,
1 ml of the above glucoamylase solution was charged to a chromatogram (φ15 x 900 mm) filled with Sephadex G-100 and developed with the same buffer.
The results are shown in FIG. A peak of glucoamylase activity was observed at the eluate volume of 65 ml. 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 operations, the specific activity based on the culture filtrate was improved 171 times. Furthermore, the activity recovery rate based on culture filtrate was 23%. Table 2 shows the specific activity, yield, and activity recovery rate of the preparation in each step based on the culture filtrate.

[Table] Note that the carbon source for 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 starch from potato, corn, sweet potato, etc., as well as soluble starch, dextrin, maltose, etc. obtained by hydrolyzing these into grapes, etc.
For maltose substrates, the rate of glucose production is approximately 1/2 that for soluble starch substrates. (3) Optimal PH Figure 2 shows the action PH curve of this enzyme at 60°C. Optimal pH of this enzyme at 60℃ or 4-5
and the preferred pH is (80% of the activity at the optimal pH)
%) is between 3.8 and 5.7. In addition, as a pH buffer during the reaction, potassium chloride-hydrochloric acid (PH2.0), glycine-hydrochloric acid (PH2.5~
3.5), β: β'-dimethylglutaric acid-trishydroxymethylaminomethane-2-amino-2
A 0.05M buffer of -methyl-1:3-propanediol (hereinafter abbreviated as "GTA") (PH3.5 to 9) was used. (4) PH stability The glucoamylase according to the present invention has a pH of 2, 3,
Under each pH of 4, 4.5, 5, 6, 7, 9 (0.05M potassium chloride-hydrochloric acid, 0.05M glycine-hydrochloric acid and
0.05 MGTA buffer) for 30 minutes at 70°C. Thereafter, the reaction solution was diluted and adjusted to pH 4.5, and residual activity was measured using soluble starch as a substrate. The result is the third
Shown in the figure. The glucoamylase according to the present invention is
The activity was completely maintained within the pH range of 4.5 to 5.0. Therefore, the present glucoamylase was found to be an enzyme having excellent stability in an acidic region. (5) Suitable temperature The activity of glucoamylase according to the present invention is expressed at PH
Measurements were made under the conditions of 4.5 at temperatures of 40, 50, 60, 65, 70, and 75°C, and the results shown in Figure 4 were obtained. From this result, the optimal temperature is around 70°C.
The preferred temperature (temperature range having 80% of the activity at the optimum temperature) is 73-73°C. (6) Effect of metal salts on glucoamylase activity Table 3 shows the effects of metal salts on the activity of glucoamylase according to the present invention. When measuring glucoamylase activity, various metal salts were added at 5mM.
It was added so that The activity was expressed in % relative to that without addition of metal salts. In addition, regarding the addition of ammonium salts and EDTA, please refer to Section 3.
Added to the table. It is recognized that magnesium ions, calcium ions, and potassium ions have glucoamylase activity. Nickel ions and iron ions have inhibitory effects.

[Table] Activity measurement conditions PH5.0 (0.1M citric acid - second sodium citrate buffer) Activity measurement temperature: 60℃ (7) Thermostability Glucoamylase according to the present invention was adjusted to PH4.5 without the addition of a substrate. Then heat treatment at 60-80℃,
At fixed intervals (20, 40 seconds, 1, 2, 4, 10, 20,
40 minutes, 1, 2, 4, 6, 8, 16 hours, 1, 2,
4, 7, 10, 15, and 20 days), a part of the treated solution was collected and the glucoamylase activity in the solution was measured at 60℃ and PH.
Measured at 4.5. Based on this, the half-life of activity at each temperature was determined, and the results are shown in FIG. The half-life of activity in the absence of substrate at 70°C and 75°C is 6 hours and 1 minute, respectively.
This glucoamylase has a higher degree of thermostability than conventionally known glucoamylases. On the other hand, the half-life of activity at 70° C. without the addition of substrate at each pH of 4.0, 4.3, 4.5, 5.0, and 6.0 was determined and shown in Table 4. Based on these results, this glucoamylase is a product of Clostridium thermoamylolidicum.
lyticum), Thermomyces lanuginosus, and Talaromyces duponti
This shows that it has superior heat resistance, especially in the acidic range of PH4 to 5, than the glucoamylase produced by.

[Table] (8) Effect of metal salts on heat resistance Table 5 shows the effects of metal salts on the heat resistance of glucoamylase according to the present invention. Add 5 m of various metal salts to an aqueous solution of glucoamylase (dissolved in 0.05 M acetic acid-sodium acetate buffer, PH4.5).
After adding the mixture to a concentration of M and heating at 70°C for 1 hour, glucoamylase activity was measured at 60°C and pH 4.5. The activity after the heat treatment, that is, the residual activity, was expressed in % compared to before the heat treatment. From Table 5, 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.

[Effect]

As is clear from the above, the new thermostable glucoamylase produced by the bacteria of the present invention is significantly better than the thermostable enzyme produced by conventional anaerobic bacteria, especially in the acidic region of PH4 to 5. different. By the way, in order to produce glucose or isomerized sugar, the raw material starch is first liquefied with α-amylase, and then saccharified with glucoamylase.
During liquefaction, the raw material starch is concentrated at a high concentration of 20 to 40%, so the pH of the liquid is acidic. For this reason, when using conventional thermostable raw α-amylase and thermostable glucoamylase, the action PH range is in the neutral range, so they must be neutralized with alkali. In contrast, the present inventors previously discovered a new heat-resistant and acid-resistant α-amylase (Unexamined Japanese Patent Publication No.
No. 236917) and the novel glucoamylase of the present invention, it is possible to perform liquefaction and saccharification in an acidic state without neutralization during liquefaction, and as a result, it is possible to carry out desalination step after the reaction. The load can be significantly reduced. [Example] Example 1 Polypeptone 0.56%, enzyme extract 0.56%, monopotassium phosphate 0.78%, dibasic sodium phosphate
0.39%, magnesium sulfate heptahydrate 0.001%,
Prepare 2000g of a synthetic medium (PH6.5) containing 0.1% sodium thioglycolate and tap water, and add 270g of it.
The mixture was dispensed into seven 500ml Sakaguchi shake flasks, and each was sterilized at 121°C for 15 minutes. On the other hand, prepare 30 g of 10% solutions of each carbon source of dextrin, potato starch, maltose, glucose, lactose, sucrose, and fructose,
Sterilization was performed at 110°C for 15 minutes. Next, the carbon source solution was added to each of the above Sakaguchi flasks in an aseptic manner. Thereafter, 30 ml of a cell suspension of Clostridium G-0005, an anaerobic bacterium of the present invention, prepared in advance using the liquid medium containing dextrin as a carbon source was added to each shaking flask. Next, after attaching a water seal trap to the gas outlet, nitrogen gas was injected into the flask to create anaerobic conditions, and the temperature was raised to 60°C.
Cultured with shaking. After 40 hours, 40 g of culture fluid was collected from each shake flask. Next, half of each culture solution sample was taken, and microbial cells were removed by centrifugation at 15,000 rpm to prepare a culture supernatant. The remaining culture fluid sample was subjected to ultrasonic disruption treatment under water cooling, and then solid matter was removed by centrifugation at 15,000 rpm to prepare a supernatant of the disrupted bacterial culture fluid. Next, the glucoamylase activity in the culture supernatant and the disrupted bacterial cell culture supernatant for each carbon source was measured. In the activity measurement, each sample was subjected to dialysis treatment using pure water for 24 hours, then heat treated at 70°C for 5 minutes, and then centrifuged to remove solid matter. The measurement results are shown in Table 6.

〔Effect of the invention〕

The novel heat-resistant/acid-resistant glucoamylase produced by the bacteria of the present invention has excellent heat resistance, especially in the acidic range of PH4 to 5. This enables saccharification in acidic conditions without oxidation. In contrast, when using conventionally known thermostable glucoamylases, an alkali must be added to neutralize the starch solution. Therefore, by using the glucoamylase produced by the bacteria 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]

FIG. 1 is a micrograph showing the biological form of the bacterium of the present invention (Clostridium genus G-0005), and FIG. 2 is a characteristic diagram showing the influence of PH on the activity of glucoamylase produced by the bacterium of the present invention. Figure 3 is a characteristic diagram showing the influence of PH on the thermostable production of the glucoamylase, Figure 4 is a characteristic diagram showing the influence of temperature on the activity of the glucoamylase, and Figure 5 is the thermostability of the glucoamylase. A characteristic diagram showing
Figure 6 is a glucoamylase activity elution pattern diagram of the glucoamylase in ion exchange liquid chromatography using diethylaminoethyl cross-linked agarose gel, and Figure 7 is the glucoamylase activity elution pattern of the glucoamylase in gel filtration using cross-linked dextran gel. It is a pattern diagram.

Claims (1)

[Claims] 1. Grows at a temperature of 49℃ to 65℃, optimal reaction temperature is 70℃, half-life of enzyme activity under heating at 70℃ at PH4.5 is 6 hours, enzyme activity under heating at 70℃ at PH4.0 A thermophilic anaerobic bacterium of Clostridium sp. No. 1455 that has the ability to produce heat-stable/acid-stable glucoamylase with a half-life of 5.7 hours.