JP2863607B2 - 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-glucoside 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] The inventors of the present invention isolated and cultured a large number of microorganisms to obtain an amylase having the above-mentioned properties, and as a result, Bacillus sp. Isolated from soil collected in Kanagawa Prefecture. It has been found that a bacterium belonging to the genus Bacillus sp. SD772 produces a novel amylase having excellent properties as a detergent enzyme, thereby completing the present invention.

That is, the present invention has 1) an optimum pH of about 10, when the 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 and a pH of 6 to 11. 3) 3000ppm
Is extremely stable to surfactants, such as having a residual activity of 95% or more even when treated at 30 ° C. for 2 hours in the presence of sodium linear alkylbenzene sulfonate (pH 9.0). 4) The molecular weight is Novel amylase having an isoelectric point of about 4.3, as measured by SDS-polyacrylamide gel electrophoresis and 5) isoelectric point as determined 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, 0.3-0.6 × 3-5μ
m, depending on the culture conditions, multiple chains are generated.

Motility Yes Spores Form round or oval swelling spores at the tips of cells.

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

Gravy agar slant culture Light yellow with rough surface. Spreading, no bumps.

Broth liquid culture medium is uniformly turbid. No coloring.

Gelatin liquefaction positive.

(C) Physiological properties Reduction of nitrate Reduces.

Denitrification reaction negative Indole formation negative Hydrogen sulfide formation negative Starch hydrolysis positive Use of citrate negative Use of inorganic nitrogen source Use nitrate and ammonium salts.

Pigment formation Negative Urease Negative Oxidase Positive Catalase Positive Range of growth (pH) In a study using nutrient broth, it grows at pH 8.0 and 9.0, but grows at pH 7.5 and 10.0
Does not grow.

(Temperature) Grows at 40 ° C but does not grow at 35 ° C. 65 ℃
It grows in but does not grow at 70 ° C.

Temperature for enzymes Aerobic OF test In the case of glucose, it does not degrade sugar. Fermentation for maltose and sucrose.

Oxidation of saccharides 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 in the presence of 7% sodium chloride Positive GC content of intracellular DNA 48% (mol%) 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.SD772 and deposited as a microbe lab. The main properties of the similar bacterium (Bacillus stearothermophilus) described in the compendium and the present bacterium are shown below.

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 a compound containing 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 target of production, are that the culture temperature is about 55 ° 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 The detailed properties 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 about 10 when the activity is measured at 30 ° C. using a soluble starch as a substrate using a Britton-Robinson broad buffer (pH 6 to 12). 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 6-11
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). FIG. 3 shows the relationship between the reaction temperature and the activity.

(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, 60
Although it was stable up to ℃ and rapidly inactivated at higher temperatures, in the presence of ₂ mM CaCl _2, 50% or more of the activity still remained after the treatment at 80 ℃. 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 about 4.3 as a result of measurement by polyacrylamide gel electrophoresis.

This enzyme is a strain belonging to the genus Bacillus bacillus sp.SD772
It has an optimum pH in the alkaline region, and has a molecular weight of 4 when compared with the thermostable alkaline amylase described in JP-A-2-49584.
While the amylase has a molecular weight of 78,00
It is clearly another enzyme by having a zero and an isoelectric point of 5.3 to 4.3.

[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 step under alkaline conditions during the refining and bleaching step, and it is possible to give a different texture 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 and aseptically dispensed into test tubes. This is Bacillus
The strain sp.SD772 was inoculated and cultured with shaking at 55 ° C. for 48 hours. The culture was centrifuged, and the amylase activity of the supernatant was measured to be 1.16 U / ml.

Example 2 3.0% peptone, 1.0% starch (soluble), wheat bran
A liquid medium consisting of 1.0%, DE-50 1.0%, yeast extract 0.5%, dipotassium hydrogen phosphate 0.5%, magnesium sulfate 0.05%, and sodium carbonate 1.0% was sterilized by a conventional method and placed in a 5 L culture tank. This was inoculated with Bacillus sp. SD772 strain that had been cultured in advance, and aerated and stirred at 55 ° C for 30 hours. This culture was centrifuged to obtain a supernatant. The amylase activity of this supernatant was 2.49 U / ml. 1.8 L of this supernatant
Was concentrated using an ultrafiltration membrane to obtain 275 ml of a 18.3 U / ml sample solution.

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

First, a precipitate of 50-70% fraction was obtained from the sample solution by an ethanol precipitation method. This precipitate was dissolved in about 200 ml of water, and a precipitate of a 40-70% fraction was obtained by an ammonium sulfate precipitation method. Further, this precipitate was dissolved in a 20 mM acetate buffer (pH 6), desalted by dialysis, and then CM equilibrated with the same buffer.
Impurities were removed using a (carboxymethyl) cation exchange resin column. The active fraction obtained by the same operation was
After dialysis against mM Tris-HCl buffer and replacing the buffer,
The mixture was adsorbed on a DEAE (diethylaminoethyl) anion exchange resin column (20 mmφ, 30 cm) equilibrated with the same buffer, and eluted with a sodium chloride concentration gradient (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, the optimal pH, pH stability, optimal temperature, thermal stability, and stability to surfactants were examined. The results 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] An amylase having the following properties: (1) Optimum pH and optimum temperature The optimum pH is about 10 and the optimum temperature is about 70 ° C. when soluble starch is used as a substrate. (2) Stable pH Stable to pH 6 to 11 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 of sodium linear alkylbenzene sulfonate (pH 9.0), it has a residual activity of 95% 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 4.3. 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.