JPS6219081A - Production of heat-resistant alpha-amylase - Google Patents

Abstract

NEW MATERIAL:alpha-Amylase having improved heat resistance and low requirement for calcium. USE:A starch modifying agent and desizing agent for textile capable of exhibiting heat resistance even when the calcium concentration is low. PREPARATION:A thermophilic anaerobic bacterium belonging to the genus Clostridium, e.g. Bacterium RS-0001 (FERM-P No.7918), is cultivated in a culture medium containing glucose polymer, preferably maltose, having two or more glucose residues as a carbon source to separate and purify the aimed enzyme secreted in the culture fluid by the well-known method.

Description

Detailed Description of the Invention [Field of Application of the Invention] The present invention relates to a method for producing a novel α-amylase using thermophilic anaerobic bacteria, and particularly to a culture medium for culturing thermophilic anaerobic bacteria. It is about quality carbon sources.

[Background of the invention]

Thermostable enzymes are more stable against heating and changes in pT than enzymes used at room temperature, and are extremely useful in enzyme utilization industries. Conventional α-amylases are limited to those originating from aerobic bacteria. (Shimamura et al.: Special Publication No. 12946/1983). Among the aerobic bacteria mentioned above, Chillus subtilis (B.
acillus 5ubtilis) and Bacillus licheniformis
α-amylase, which originates from B.formls), has already been produced industrially and is used for starch processing and fiber desizing. All of these known α-amylases cannot exhibit heat resistance only with the protein of the enzyme itself, and only show heat resistance in the presence of calcium ions. Usually several mm
A calcium concentration of ~20mM (Hanbu: JP-A-51-44652, JP-A-51-44690) and at least 1mM (Saito: JP-A-48-35083) is required.

The present inventors searched for enzymes and enzyme-producing microorganisms with the aim of obtaining α-amylase with excellent heat resistance and low calcium requirement. As a result, obligate anaerobic bacteria belonging to the genus Clostridium (Clostridium RS-0001゜clostridium sp.
RS-0001, Microtech Research Institute No. 7918) discovered that a new thermostable α-amylase was produced whose enzyme properties, especially calcium requirement and action pH range, were completely different from conventional α-amylases. .

The production of α-amylase involves (i) culturing bacteria using starch, dextrin, etc. as a carbon source, and adding α-amylase to the culture solution.
secretes amylase. (ii) α-
Collect and purify amylase. It is carried out according to the following steps. Therefore, in order to efficiently produce α-amylase, it is necessary to secrete α-amylase efficiently into the culture solution.

[Purpose of the invention]

The purpose of the present invention is to produce thermophilic anaerobic bacteria that can secrete a large amount of a novel α-amylase with excellent heat resistance and low calcium requirement into the culture solution. The objective is to provide a carbon source for cultivation.

[Summary of the invention]

The present inventors searched for enzyme-producing microorganisms with the aim of obtaining a new type of α-amylase with excellent heat resistance and low calcium requirement. As a result, thermophilic anaerobic bacteria belonging to the genus Clostridia (R8-0001, C1ostridium
Ilsp, RS-0001, Microtechnical Research Institute No. 7918
No. 2) was found to produce a new thermostable α-amylase that satisfies the above requirements. Then, using this bacterium, α
- As a result of intensive research into an efficient method for producing amylase, the present invention was achieved.

A feature of the present invention is that maltose is used as a carbon source in the culture medium used when culturing bacteria belonging to the genus Clostridium. By using maltose as a carbon source, compared to using starch or dextrin,
The amount of α-amylase secreted into the culture solution reaches about twice as much. Moreover, maltose promotes the secretion of α-amylase not only when used alone as a carbon source but also when starch or soluble starch and maltose are used in combination. That is, a larger amount of α-amylase is secreted into the culture solution than when starch or soluble starch or dextrin is used alone as a carbon source.

Note that, like maltose, in the case of isomaltose, which is a condensation product of two glucose molecules, α-
No amylase production promoting effect was observed.

α-Amylase secreted into the culture solution can be detected using conventional methods such as adsorption onto starch particles, salting out using ammonium sulfate, filtration using a molecular sieve membrane, molecular sieve chromatography, and ion exchange liquid chromatography. After separation and purification by a method known as amylase purification method, it is commercialized.

Bacteria belonging to the genus Clostridium (Clostridium spR) that produce α-amylase used in the present invention
S-0001) has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology (accession number: Microbiological Research Institute No. 7918 (FE
RM P-7918)). First, the details of the mycological properties of this bacterium will be explained.

A. Morphological properties (1) Morphology of vegetative cells When cultured on an agar plate in the following starch/peptone medium in an anaerobic atmosphere at 60°C for 2 days, vegetative cells have a
It is a straight bacillus with a size of 0.8 x 2 to 5 μm. When cultured for 3 days or more, vegetative cells with the above-mentioned shape exist singly, and vegetative cells in chains also occur. The same holds true for liquid culture.

Composition of starch/peptone medium Soluble starch 1.5% peptone
0.5% yeast extract 0.5%KH, PO40
,7% Na2HP0. 0.35%MgS
O-・7 H200-001% agar
2.0% Sodium thioglycolate 0.1
% tap water pH 6.4 (2) Presence or absence of spores Spore formation is observed in agar plate culture and liquid culture in starch/peptone medium.

B. Culture characteristics (1) Colony morphology Colonies in agar plate culture on starch/peptone medium have a flat, circular shape with a slightly raised center, and a golden edge at the periphery. Pigment formation is visible, and the surface is opalescent and opaque with photosensitivity. It also has adhesive properties.

(2) Agar plate culture and puncture feeding on meat juice medium produce colonies similar to those on starch/peptone medium.

Meat juice agar medium composition Meat extract 1.0% peptone
1.0% salt
0.2% Sodium thioglycolate 0.1% Agar
1.5% distilled water pH 6.0 (3) Puncture culture of meat broth culture Grows with the generation of gas containing hydrogen and carbon dioxide, and as a result, the agar medium is divided into two or three places.

(4) Meat juice liquid culture It grows only in an anaerobic atmosphere, and the culture solution becomes cloudy.

Composition of meat juice medium Meat extract 1.0% peptone
1.0% salt
0.2% Sodium thioglycolate 0.1% Distilled water pH 6,0 (5) No growth of meat juice/gelatin culture was observed.

Composition of meat juice/gelatin medium Meat extract 1.0% peptone
1.0% salt
0.2% gelatin 15% sodium thioglycolate 0.1% distilled water pH 6.0 (6) Lidomus milk culture Coagulates solidly with gas generation and turns red due to acid production.

C0 Physiological properties (1) Growth temperature range: Grows at 40-63°C. No growth was observed at 30℃,
Good at around 60℃.

(2) PH range for growth pH around 5-7°5.6 is good.

(3) Obligate anaerobic attitude toward oxygen (4) 〇-F test (Hugh Laj, fson modified method) No growth was observed in air atmosphere, negative. Bacteria grow under the anaerobic conditions of the liquid paraffin overlay, producing acid and turning the culture solution yellow.

Medium composition peptone 0.2% glucose
1.0% salt
0.5% 2HPO40.03% Sodium thioglycolate 0.1% Bromucresol Purple 0.002% Agar
0.3% distilled water pH 6,0 (5) Nitrate reduction negative.

(6) VP test negative.

(7) MR test Positive, turns red.

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

(9) Generation of hydrogen sulfide Negative (in using Kligrer's medium).

(10) Starch hydrolysis positive. It breaks down not only soluble starches, but also granular starches such as potato starch.

(11) Use of citric acid Negative (using Simn+ons medium).

(12) Use of ammonium salt It cannot be measured because it does not grow in peptone water.

(13) Extracellular production of pigment negative.

(14) Urease negative.

(15) Oxidase activity negative.

(16) Catalase activity negative.

(17) Sugar assimilation Me assimilation of sugar and presence or absence of gas generation using Durham tube
The results are shown in the table below.

Chapter 1 Table (I8) Growth on mineral salt medium Not recognized as growing.

(19) Generation of organic acids Table 2 shows the composition of organic acids produced from various media.

Table 2 Composition of sample liquid medium Carbon source 1.0% peptone
1.0% salt
0.2% Sodium thioglycolate 0.1 Distilled water pH 6.4 From these results, it was identified as a bacterium belonging to the genus Clostridium based on Holdeman's anaerobic bacteria classification manual.

The α-amylase activity was measured as follows.

Blue value method (edited by the Chemical Society of Japan: Experimental Chemistry Course Volume 24, Biochemistry ■, p279,
The glue refining power was measured according to the method (Maruzen Shoten, 1969). This method applies the principle that as the starch molecules are hydrolyzed, the amount of blue color produced by the starch-more elementary complex decreases in proportion to the decrease in molecular weight. be. First, 2 mA of a 2 mg/m Q starch solution and 1 mΩ of 0.1 M < citric acid buffer (pH 4,0) were placed in a test tube and shaken for 5 minutes in a 60°C water bath. Then,
1 mρ of culture furnace solution was added as a crude oxygen solution, and the mixture was allowed to react for 30 minutes. After the reaction, 0.4 mQ of the reaction solution was collected and immediately diluted with 0.4 mQ.
The enzyme reaction was stopped by mixing with 2 m Q of 5M acetic acid solution. The next 1 mfl is 10 m (1/300 of l)
It was added to an iodine solution, and the absorbance at 680 nm was measured using a spectrophotometer.

On the other hand, the reaction solution immediately after adding the enzyme solution (hereinafter referred to as 01 reaction solution) was sampled, colored in the same manner, and the absorbance was measured. Note that amylose with a degree of polymerization of about 2000 was used as the starch.

α-amylase activity was calculated using the following formula.

α-amylase activity (units) = Absorbance of Ot reaction solution [Embodiments of the invention] Hereinafter, examples of the present invention will be shown and explained in detail.

Example 1 Enzyme extract 0.5%, polypeptone 0.5%, monopotassium phosphate 0.7%, dibasic sodium phosphate 0.2%,
Magnesium sulfate heptahydrate o, oot%, sodium thioglycolate 0.1%, maltose 2 as carbon source
%, and a liquid medium containing tap water (pH 6,0) 2.
Put 7 kg into a culture tank with an internal volume of 5Q and heat at 120℃ for 1 hour.
Sterilized for 0 minutes. To this was added 0.3 kg of a cell suspension of Clostridium bacteria that had been anaerobically cultured in the same 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, followed by culturing under anaerobic conditions. The pH of the culture solution is automatically adjusted to 6.0, and the temperature is also 60.
Automatically adjusted to ℃. During the culture, a portion of the culture solution in the tank was collected at regular intervals and centrifuged at 3500 ppm.
After sterilization by filtration with a 0.2 μm filter, α-amylase activity in the liquid was measured. As a result, as shown by curve 1 in FIG. 1, a maximum of 61 units/mQ of α-amylase was produced 16 hours after the start of culture.

Comparative Example 1 In Example 1, a culture test was conducted in the same manner as in Example 1, using 2% potato starch as a carbon source. As a result of measuring the production amount of α-amylase, as shown in curve 2,
It was 28 units/mQ 44 hours after the start of culture.

Comparative Example 2 In Example 1, a culture test was conducted in the same manner as in Example 1, using 2% soluble starch as a carbon source. As a result of measuring the production amount of α-amylase, as shown in curve 3, it was 30 units/mQ 40 hours after the start of culture.

Comparative Example 3 In Example 1, a culture test was carried out in the same manner as in Example 1, using 2% dextrin as a carbon source. When the production amount of α-amylase was measured, as shown in curve 4,
It was 30 units/mQ at 38 hours from the start of culture.

Comparative Example 4 In Example 1, a culture test was carried out in the same manner as in Example 1, using glucose as a carbon source. As a result of measuring the production amount of α-amylase, as shown in curve 5, it was 6 units/mQ 60 hours after the start of culture.

Comparative Example 5 In Example 1, a culture test was carried out in the same manner as in Example 1, using isomaltose as a carbon source.

As a result of measuring the amount of α-amylase produced, as shown in curve 6, it was 15 units/mQ 60 hours after the start of culture.

By comparing Example 1 and Comparative Examples 1 to 5, it can be seen that by using maltose as a substrate, high concentration α-amylase can be produced in a short time culture.

Example 2 In Example 1, a culture test was conducted by adding 1.5% starch and 0.5% maltose as carbon sources. The amount of α-amylase produced reached 52 units/mD 46 hours after the start of culture, as shown by curve 11 in FIG.

Example 3 In Example 1, 1.5% soluble starch was used as a carbon source.
And a culture test was conducted by adding 0.5% maltose.
As shown in curve 12, the α-amylase production amount reached 56 units/mQ 45 hours after the start of culture.

Example 4 In [Example], 1.5% dextrin was used as a carbon source.
A culture test was conducted by adding 0.5% maltose.

As shown in curve 13, the α-amylase production amount reached 58 units/mQ 41 hours after the start of culture.

Comparing Example 2 and Comparative Example 2, comparing Example 3 and Comparative Example 3, and comparing Example 4 and Comparative Example 4, it was found that maltose is not only used as a carbon source alone, but also when maltose is used as a carbon source alone. It can be seen that by mixing with each of starch, soluble starch, and dextrin, the amount of α-amylase produced increases compared to when each of these is used alone.

Example 5 Potato starch 2%, polypeptone 0.5%, yeast extract 0
．． 5%, potassium phosphate 0.7%, dibasic sodium phosphate 0.2%, magnesium sulfate heptahydrate 0.00
1%, sodium thioglycolate 0.1% and tap water (pH 6,0) 2.7 kg to 90
℃ for 5 minutes, and then cooled to 60°C. Next, 1000 units of β-amylase was added to this,
The reaction was carried out at 60°C and pH 6.0 for 1 hour. After 1 hour, the reaction solution contained 0.54% maltose. Next, this was placed in a culture tank with an internal volume of 5Q and incubated at 120°C for 1 hour.
High-pressure steam sterilization was performed for 0 minutes. A culture test was conducted in the same manner as in Example 1, and 56 units/mQ of α-amylase was produced 38 hours after the start of culture.

After the culture was completed, the cells were cooled to 4° C. and then centrifuged at 7000 rpm to remove the bacterial cells. Then, the pore size is 0
．． The culture solution was clarified using a 45 μm filter to obtain 2.8 kg of cultured solution. Next, 150 g of corn starch was added while cooling to 4° C., and after contacting with stirring for 10 minutes, centrifugation was performed to collect corn starch. Pure water IQ cooled to 4° C. was added to the collected starch to wash the starch. Next, 0.8% pure water heated to 65°C
The above corn starch was added to Q to desorb α-amylase. Immediately thereafter, starch particles were removed using a centrifugal filter to obtain 0.75 Q of α-amylase solution.

The α-amylase solution was concentrated using a molecular sieve membrane (molecular weight cut off: 20,000) and then freeze-dried.
5 mg was obtained.

〔Effect of the invention〕

As detailed above, when producing thermostable α-amylase using thermophilic anaerobic bacteria of the genus Clostridia,
By applying the present invention and using a mixture of glucose polymers having two or more glucose residues as a carbon source in the culture medium, α-amylase with excellent heat resistance and low calcium requirement can be produced in the culture medium. It has an excellent practical effect of being able to secrete a large amount into the body.

[Brief explanation of drawings]

FIGS. 1 and 2 are charts for explaining the effects of the embodiments of the method of the present invention, respectively. 1.11,12,13...α in the embodiment of the present invention
- a curve showing the change in amylase activity over time, 2,
3, 4.5.6... Comparative Example 1 Curve showing changes in α-amylase activity over time. Agent Patent Attorney I: Akimoto 1 [: Actual 1 Figure 1 Sai! between u〒(h)

Claims (1)

[Claims] 1. When thermostable α-amylase is produced using thermophilic anaerobic bacteria belonging to the genus Clostridium, the culture substrate for culturing the thermophilic anaerobic bacteria contains a carbon source as a carbon source. 1. A method for producing heat-stable α-amylase, which comprises using a mixture of glucose multimers having two or more glucose residues. 2. The method for producing thermostable α-amylase according to claim 1, wherein the glucose multimer is a mixture of two or more types of glucose multimers. 3. The method for producing thermostable α-amylase according to claim 1, wherein the glucose multimer is maltose, which is a glucose dimer. 4. The method for producing heat-stable α-amylase according to claim 1, wherein the glucose multimer is maltose and is used in combination with starch. 5. The heat-stable α-amylase according to claim 4, wherein the carbon source is a mixture of starch as a main carbon source and maltose as a secondary carbon source. manufacturing method.