JP2863602B2 - Amylase and production method thereof - Google Patents

Description

The present invention relates to a novel amylase, a method for producing the same, and an amylase-producing microorganism.

[Prior Art] Amylase is a hydrolase that acts on starch or a partially degraded product thereof to decompose α-1,4-glycosidic bonds. Industrially, it is widely used in starch processing, food processing, textile processing, brewing, pharmaceuticals, clinical tests, etc.
Its origins also range from microorganisms, plants and animals.

Another use of amylase has been shown to be useful in detergent formulations. Concretely, it can be used in laundry detergents, detergents for automatic dishwashers, etc., but considering the conditions during use, it is desirable to have an optimum pH for alkali and excellent stability to heat. It is. However, all enzymes used industrially at present are neutral amylase, and there is no enzyme that is suitable for blending in detergents. Accordingly, all detergent-combined enzymes that are already commercially available are neutral amylases, and there is no problem in terms of heat resistance, but it is hard to say that they are functioning sufficiently.

[Problems to be Solved by the Invention] An object of the present invention is to provide an amylase having an optimum pH on the alkaline side and sufficiently maintaining the activity even at a high temperature and in the presence of a surfactant.

[Means for Solving the Problems] As a result of separating and culturing a large number of microorganisms in order to obtain an amylase having the above properties, the present inventors found that the soil in Kumamoto Prefecture was 1% sodium carbonate, % Potato starch, and allowed to stand at 65 ° C. for 2 weeks to 6 weeks, and isolated after being isolated from a bacterium belonging to the genus Bacillus sp.
The strain was found to produce a novel amylase having excellent properties as a detergent enzyme, and the present invention was completed.

That is, the present invention provides 1) an optimum pH of 9 to 11 when soluble starch is used as a substrate, an optimum temperature of about 70 ° C., and 2) a stable pH range of 50 ° C. for 30 minutes when the pH is 6 to 12. Yes, 3) 3000p
Extremely stable to surfactants, such as having 90% or more residual activity even after treatment at 30 ° C for 2 hours in the presence of sodium linear alkyl benzene sulfonate (pH 9.0) at pm. 4) Molecular weight Is a 45,000 as determined by SDS-polyacrylamide gel electrophoresis, and 5) a novel amylase having an isoelectric point of 4.25 as measured by polyacrylamide gel electrophoresis; A method for producing the above-mentioned amylase, which comprises culturing a microorganism in a medium and collecting a target amylase from the culture; and a novel strain of the genus Bacillus having the above-mentioned amylase-producing ability.

Hereinafter, the amylase of the present invention, a method for producing the amylase, and a novel strain used therein will be described in more detail.

Producing Bacteria The microorganism used for producing the amylase of the present invention is a bacterium belonging to the genus Bacillus capable of producing the amylase having the above-mentioned properties, and has the following properties.

(A) Shape Cell shape and size Bacillus, 0.3-0.7 × 3-6μ
m, depending on the culture conditions, multiple chains are generated.

Motility Yes Spores Form round, swollen spores at the tips of cells.

Gram stain positive (b) Growth conditions in each medium Broth agar plate culture Colonies are pale yellow and translucent.
Circular, flat and wavy.

Gravy agar slope culture Grows but is not very good.

Liquid broth culture Form precipitates of bacterial cells on the bottom. The medium is neither turbid nor colored.

Gelatin liquefaction negative.

(C) Physiological properties Reduction of nitrate Not reduced.

VP test Negative Starch hydrolysis Positive citrate utilization Negative Catalase positive Growth range (pH) Grows at 7.5 but not at 6.8.

 Grow at 10.3.

(Temperature) Grows at 50 ° C but does not grow at 40 ° C.
Grows at 70 ° C but does not grow at 71 ° C.

Attitude to enzymes Oxidation of aerobic sugars and generation of gas Oxidized gas (1) L-arabinose ±-(2) D-xylose ±-(3) D-glucose ±-(4) D-mannose ±-( 5) D-fructose ± − (6) D-galactose ± − (7) Maltose ± − (8) Sucrose − − (9) Lactose ± − (10) Trehalose − − (11) D-Sorbit − − (12 ) D-mannitol--(13) Inosit--(14) Glycerin--(15) Starch +-Growth negative in the presence of 5% sodium chloride Negative microbiology with a GC content of intracellular DNA of 48% (mol%) or more This gold was identified as a bacterium belonging to the genus Bacillus. However, this bacterium is a bacterium that grows only at high temperatures and on the alkaline side, and a description of strains exhibiting these characteristics is given in Bergey's Manual of Systematic Bacteriology (Bergey's Manual of Systematic Bacteriology).
tic Bacteriology) and the Genus Bacillus (A Gena Bacillus USDA Handbook No. 427). Therefore, the strain was named Bacillus sp.SD771 and deposited as Microbial Laboratory No. 11455. The main differences between the similar bacteria described in the compendium and the present bacteria are shown below.

(1) Bacteria that grow only on the alkaline side.

(2) Bacteria that grow only on the high temperature side.

Culture method In producing the amylase of the present invention, the culture medium for culturing the above-mentioned microorganisms does not need to be exceptional, and nutrient sources commonly used for culture can be widely used. The carbon source may be any assimilable carbon compound or a compound containing the same. For example, glucose, maltose, starch and the like are used. The nitrogen source may be any assimilable nitrogen compound or any compound having the same. For example, peptone, soybean powder, CSL and the like are used. Salts such as phosphates and magnesium salts are used as the inorganic salts. In addition, various organic and inorganic substances necessary for the growth of bacteria and production of enzymes, or those containing these substances, such as yeast extract and vitamins, can be added to the medium. Culture is preferably liquid culture,
Culture conditions vary depending on the medium composition. The most advantageous conditions for the production of amylase, which is the object of production, are that the culture temperature is about 60 ° C under aerobic conditions, the culture time is about 12 hours to 3 days, and the production of amylase is the highest. The culture may be terminated when it has been reached. The pH of the medium is preferably 8 or more, and 9.5 to 1
0 is suitable for amylase production. The amylase of the present invention is mainly secreted and accumulated in the culture solution.

Isolation and Purification Method Amylase can be collected from the obtained culture solution by separation and purification according to a conventional method for obtaining an enzyme. By a suitable method such as filtration and centrifugation, a solid substance such as cells in the medium can be separated to obtain a supernatant.
Amylase can be obtained by concentrating the separated liquid, spray drying, freeze drying, salting out, precipitation with a hydrophilic organic solvent, or the like. Further, in order to purify the enzyme, there is a method in which a purification means such as an ion exchange resin and a gel filtration method is used alone or in combination.

Enzyme Properties A detailed description of the amylase of the present invention will be described.

1. Method for measuring enzyme titer.

The measurement is performed using a 1% aqueous solution of soluble starch as a substrate. Specifically, 0.5 ml of the substrate is added to 0.5 ml of the buffer solution, incubated at a predetermined temperature for several minutes, and 0.05 ml of the enzyme solution is added to start the reaction. After 10 minutes, the reaction is stopped by adding 1 ml of 1N hydrochloric acid. Aliquot 0.4 ml from the solution that has stopped the reaction, 5 ml
Dilute with water. 0.5% iodine-potassium iodide solution
After 0.125 ml is added for color development, colorimetry is performed at a wavelength of 700 nm. The enzyme titer was defined as 0.1 unit, which is the amount of the enzyme which reduces the absorbance of the unreacted starch and iodine by 10% corresponding to 10% of the color in one minute under the above conditions.

2. Properties of the enzyme (1) Action The enzyme acts on starch or its partially decomposed product to hydrolyze α-1,4-glycosidic bonds, and mainly produces dextrin and maltooligosaccharide. Also,
The decomposition is end-type.

(2) Optimum pH The activity is 9 to 11 when the activity is measured at 30 ° C. using Britton-Robinson's wide-range buffer (pH 6 to 12) and soluble starch as a substrate. FIG. 1 shows the relationship between the reaction pH and the activity.

(3) Stable pH range After using Britton-Robinson's wide-range buffer (pH 3 to 12) at 50 ° C for 30 minutes at each pH, the activity at 30 ° C and pH10 was measured. Stable pH is 6-12
It is. FIG. 2 shows the relationship between the treatment pH and the residual activity.

(4) Optimal temperature The optimal temperature is about 70 ° C. when measured using a 50 mM CHES buffer (pH 9). Relationship between reaction temperature and activity
Shown in the figure.

(5) Thermal stability Under 50 mM CHES buffer (pH 9), each temperature (30 ° C to 80 ° C)
° C) for 10 minutes, and after cooling on ice, the residual activity at 30 ° C was measured. In the presence of 2 mM ethylenediaminetetraacetic acid, 5
It is stable up to 0 ° C and rapidly deactivated at higher temperatures, whereas in the presence of ₂ mM CaCl _2, 50% or more of the activity still remains after the treatment at 80 ° C. FIG. 4 shows the relationship between the heat treatment temperature and the residual activity.

(6) Influence of surfactant.

Sodium linear alkylbenzene sulfonate 3000ppm
Almost no inactivation occurs when treated at 30 ° C for 2 hours in the presence (pH 9). FIG. 5 shows the relationship between the treatment time and the residual activity.

(7) Molecular weight The molecular weight obtained by SDS-polyacrylamide gel electrophoresis is about 45,000.

(8) Isoelectric point The isoelectric point was 4.25 as measured by polyacrylamide gel electrophoresis.

This enzyme is a strain belonging to the genus Bacillus Bacillus sp.SD7
It is produced by strain 71 and has an optimum pH in the alkaline region, and when compared with the thermostable alkaline amylase described in JP-A-2-49584, the molecular weight of the enzyme is
While the molecular weight of the amylase is 78,0
It is clearly another enzyme because of its 00 and isoelectric point of 5.3 to 4.25.

[Effect of the Invention] The present enzyme is an enzyme showing high activity in a high temperature and alkaline region. At present, all of the amylase that acts at high temperatures are neutral enzymes, and alkaline enzymes are weak to heat. Enzymes satisfying both are not yet commercially available. By using this enzyme, starch degradation in a high-temperature alkaline region can be significantly improved. For example, in the textile industry, it can be used in the desizing process under alkaline conditions during the scouring and bleaching process, and it becomes possible to give a texture different from the conventional one. In addition, since it has high stability against an anionic surfactant as a detergent component, it can be used as a detergent enzyme, and the detergency can be enhanced. Furthermore,
By blending it in detergent for automatic dishwashers, it is possible to improve the cleaning effect on starchy stains such as rice grains. In addition to this, it can be used in a very wide range of fields such as a step of processing starch in an alkaline region.

Next, the present invention will be described more specifically with reference to typical examples.

Example 1 Peptone 2%, Starch (soluble) 2%, Yeast extract 0.
1%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.
A liquid medium consisting of 02% and 1% sodium carbonate was sterilized by an ordinary method, dispensed aseptically into test tubes, inoculated with strain SD771, and cultured with shaking at 60 ° C for 48 hours. The culture was centrifuged, and the amylase activity of the supernatant was measured.
U / ml.

Example 2 2.5% peptone, 2.5% casamino acid, starch (soluble)
A liquid medium consisting of 2.5%, DE-50 2.5%, yeast extract 0.5%, dipotassium hydrogen phosphate 0.5%, magnesium sulfate 0.05%, calcium carbonate 0.05%, sodium carbonate 0.8% is sterilized by a conventional method, and a 5L culture tank is prepared. Put in. The SD771 strain which had been cultured beforehand was inoculated into this, and aeration and agitation culture was performed at 60 ° C. for 50 hours. This culture was centrifuged to obtain a supernatant. The amylase activity of this supernatant was 0.65 U / ml.
2.2 L of the supernatant was concentrated with an ultrafiltration membrane to obtain 300 ml of a 4.75 U / ml sample solution.

Example 3 The amylase of the present invention was purified from the sample solution obtained in Example 2.

First, from the sample solution, 50-
A precipitate of 70% fraction was obtained. This precipitate was dissolved in a 20 mM acetate buffer (pH 6), desalted by dialysis, and impurities were removed by a CM (carboxymethyl) cation exchange resin column equilibrated with the same buffer. The active fraction obtained by the same operation was dialyzed against a 20 mM Tris-HCl buffer, the buffer was replaced, and then a DEAE (diethylaminoethyl) anion exchange resin column (20 mmφ, 30 cm) equilibrated with the buffer was used. And eluted with a concentration gradient of sodium chloride (0-1 M). The obtained active fraction (50 ml) was concentrated to 2 ml by ultrafiltration, and subjected to gel filtration using Sephadex G-75 (25 mmφ, 90 cm). The activity was detected as two peaks, and it was confirmed that the rear peak was an electrophoretically nearly uniform sample. Using this purified sample, optimal pH, pH
Stability, optimum temperature, thermal stability, and stability to surfactant are shown in FIGS. 1 to 5.

[Brief description of the drawings]

FIG. 1 is a graph showing the relationship between reaction pH and activity. FIG. 2 is a graph showing treatment pH and residual activity. FIG. 3 is a graph showing the relationship between reaction temperature and activity. FIG. 4 is a graph showing the relationship between the heat treatment temperature and the residual activity. Figure 5 shows sodium linear alkylbenzene sulfonate
4 is a graph showing stability in the presence of 3000 ppm.

──────────────────────────────────────────────────の Continued on the front page (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 9/28 BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. Amylase having the following properties: (1) Optimum pH, optimum temperature The optimum pH is 9 to 11 and the optimum temperature is about 70 ° C. when soluble starch is used as a substrate. (2) Stable pH Stable to pH 6 to 12 when treated at 50 ° C for 30 minutes. (3) Influence of surfactant Even when treated at 30 ° C. for 2 hours in the presence of 3000 ppm sodium linear alkylbenzene sulfonate (pH 9.0), it has a residual activity of 90% or more. (4) Molecular weight The molecular weight measured by SDS-polyacrylamide gel electrophoresis is about 45,000. (5) Isoelectric point The isoelectric point measured by isoelectric focusing is about 4.25. 2. The amylase according to claim 1, wherein the microorganism belonging to the genus Bacillus and having the amylase-producing ability according to claim 1 is cultured in a medium, and the target amylase is collected from the culture. Production method.