JPH0856662A - Amylase, its production and microbe capable of producing the same - Google Patents

Abstract

PURPOSE: To obtain a new amylase presenting high activity at elevated temperatures in alkaline region, capable of hydrolyzing the α-1,4-glucoside linkage of starch, thus useful as a detergent enzyme or for starch processing, by culturing Bacillus bacteria. CONSTITUTION: This new amylase is obtained by culturing bacteria belonging to Bacillus and having ability to produce amylase [e.g. Bacillus sp. SD 773 strain (FERM P-14462)] and taking the resultant amylase from the cultured product. This amylase has the following characteristics: activity: hydrolyzing the α-1,4-glycoside linkage of starch by acting thereon; optimal pH: about 10.5 when reacted at 60 deg.C for 10min with soluble starch as substrate, and optimal temperature: about 65 deg.C when reacted at pH10 for 10min; stability: stable at pH6-11 when treated at 40 deg.C for 30min, having 100% residual activity up to 60 deg.C in the presence of 2-mM CaCl2 when treated at pH10 for 30min, while 60% residual activity at 50 deg.C in the absence of 2mM CaCl2 ; and molecular weight: 110000 (SDS-PAGE).

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel amylase, a method for producing the same, and a microorganism producing the amylase.

[0002]

BACKGROUND OF THE INVENTION Amylase is widely used industrially for starch processing, food processing, fiber processing, brewing, medicine, clinical examinations, etc., and its origins are diverse including microorganisms, plants and animals. Another application of amylase has been shown to be useful in detergent formulations. Specific examples include blending with laundry detergents and detergents for automatic dishwashers. Considering the conditions during use, it is desirable that they have an optimum pH for alkalis and excellent stability against heat. Be done. However, all of the enzymes currently used industrially are acidic or neutral amylase, and there is no enzyme that exerts a sufficient function under actual washing conditions.

[0003]

DISCLOSURE OF THE INVENTION The present invention is to provide an amylase having an optimum pH on the alkaline side and having sufficient activity even at high temperatures.

[0004]

[Means for Solving the Problems] The present inventors have conducted a search by isolating and culturing a large number of microorganisms to obtain an amylase having the above-mentioned properties. As a result, the genus Bacillus isolated from soil collected under Hyogo Prefecture It was found that the Bacillus bacillus sp. SD773 strain belonging to the group produces a new amylase having excellent properties as an enzyme for detergent, and has completed the present invention.

DISCLOSURE OF THE INVENTION The present invention provides the following novel amylase, a method for producing the same, and a new strain. 1) An amylase having the following properties. (1) Action It acts on starch to hydrolyze the α-1,4-glycoside bond. (2) Optimum pH When a soluble starch is used as a substrate and reacted at 60 ° C. for 10 minutes, the optimum pH is about 10.5. (3) Optimum temperature When soluble starch is used as a substrate and reacted at pH 10 for 10 minutes, the optimum temperature is about 65 ° C. (4) Stable pH When treated at 40 ° C for 30 minutes, it is stable in the pH range of 6-11. (5) Thermal stability When treated at pH 10 for 30 minutes, it has a residual activity of 100% up to 60 ° C in the presence of 2 mM calcium chloride, and a residual activity of about 60% at 50 ° C in the absence thereof. (6) Molecular weight The molecular weight measured by SDS-polyacrylamide gel electrophoresis is about 110,000.

2) An amylase having the following properties. (1) Action It acts on starch to hydrolyze the α-1,4-glycoside bond. (2) Optimum pH When a soluble starch is used as a substrate and reacted at 60 ° C. for 10 minutes, the optimum pH is about 10. (3) Optimum temperature When soluble starch is used as a substrate and reacted at pH 10 for 10 minutes, the optimum temperature is about 65 ° C. (4) Stable pH When treated at 40 ° C for 30 minutes, it is stable in the pH range of 6-11. (5) Thermal stability When treated at pH 10 for 30 minutes, it has a residual activity of about 90% at 60 ° C in the presence of 2 mM calcium chloride, and a residual activity of about 60% at 50 ° C in the absence. (6) Molecular weight The molecular weight measured by SDS-polyacrylamide gel electrophoresis is about 90,000.

3) A method for producing amylase, which comprises culturing a bacterium belonging to the genus Bacillus having the ability to produce amylase described in 1) above and collecting the amylase from the culture. 4) A method for producing amylase, which comprises culturing a bacterium of the genus Bacillus having the ability to produce amylase according to 2) above and collecting the amylase from the culture. 5) The bacterium of the genus Bacillus is Bacillus sp. SD773 strain (F
The method for producing amylase according to the above 3) or 4), which is ERM P-14462) or a mutant strain or genetically engineered strain thereof. 6) A bacterium belonging to the genus Bacillus having the amylase-producing ability according to 1) or 2) above. 7) Bacillus sp. SD773 strain (FERM P-14
462) or a mutant or genetically engineered strain thereof.

The amylase of the present invention, the method for producing the same, and the novel strains used therein will be described in more detail below.

(Amylase-Producing Bacterium) The microorganism used for producing the amylase of the present invention may be any bacterium capable of producing the amylase, preferably Bacillus bacterium, more preferably Bacillus sp. The SD773 strain or its mutant strain or genetically engineered strain.

Below, Bacillus sp. The mycological property of SD773 strain is shown. (A) Morphology Cell shape and size; bacillus, 0.7 × 3 to 4.5 μm, producing chains depending on culture conditions. Motile or not; yes Spores: Form a rounded, slightly swollen sporangium in the center of the cell. Gram stain; Positive (b) Growth condition in each medium Nutrient broth Grows. Glucose-Nutrient Broth Grows. Aerobic culture in glucose broth Grows. Anaerobic culture in glucose broth Grows. (C) Physiological properties Gelatin liquefaction Positive hydrolysis of casein Positive hydrolysis of starch Positive Use of citrate Positive catalase Positive VP test Negative Growth range (pH) 6.0 to 10.0. (Temperature) It grows up to 45 ℃. Acid production from sugars (1) D-glucose Does not grow. (2) D-maltose Does not grow. (3) D-arabinose Does not grow. (4) D-lactose Does not grow. (5) Does not form D-mannitol acid. It grows with a salt concentration of 2% in nutrient broth. It grows 10%.

This strain has been deposited as FERM P-14462 at the Institute of Biotechnology, Institute of Biotechnology, AIST.
This bacterium was identified as a bacterium belonging to the genus Bacillus based on the above-mentioned mycological properties. And the Burgies Manual of
Systematic Bacteriology (Bergey's Manual o
f Systematic Bacteriology) and similar strains described in The Genus Bacillus USDA Handbook No. 427 and main differences between the present strains are shown below. Bacillus megaterium The size of this bacterium 1.0 μm or more 0.7 μm Growth under anaerobic conditions No growth. To grow.

From the above-mentioned mycological properties, the present bacterium is a novel strain which is clearly different from other known strains. Moreover, a mutant strain producing the amylase of the present invention can be obtained by natural or induced mutation using the present bacterium as a progenitor strain and can be used as the amylase-producing bacterium of the present invention.
As a method for preparing these mutant strains, for example, as a conventional method, the original strain is not subjected to artificial mutation treatment, or is subjected to ultraviolet irradiation treatment or N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or the like. By applying an artificial mutation treatment with a drug, spread on an agar medium containing soluble starch etc., the size of the clear zone formed around the colony from the growing strains, select a larger colony, There is a method of culturing in an amylase-producing medium to select a strain having the highest productivity.

As a method for preparing a genetically engineered strain that can be used as the amylase-producing bacterium of the present invention, a conventional method can be used. For example, the DNA base sequence is deduced from the amino acid sequence of amylase, the DNA of the characteristic portion of the sequence is synthesized, and the phosphorus atom of the phosphate group is labeled with radioisotope 32P. On the other hand, whole chromosomal DNA is extracted from the original strain and cleaved with an appropriate restriction enzyme,
Synthetic DNA labeled above and Southern Hybridyzation
A fragment of chromosomal DNA that hybridizes is selected by the method. Chromosomal DNA selected in this way
The above fragment is incorporated into an appropriate vector and introduced into an amylase non-producing strain to check the presence or absence of amylase production. By introducing a DNA fragment whose amylase production has been confirmed into an original strain or a strain having high enzyme productivity (high protein secreting ability) using an appropriate vector such as a plasmid, a strain having improved productivity can be obtained.

(Bacterial Cultivation) In producing the amylase of the present invention, the medium for culturing the above-mentioned microorganism does not need to be special, and those commonly used for culturing can be widely used as nutrient sources. . As the carbon source, any carbon compound that can be used or one that contains it can be used.
For example, glucose, maltose, starch, etc. are used. Any nitrogen compound that can be used as a nitrogen source or a compound containing the same may be used, and for example, peptone, soybean powder, CSL (corn steep liquor), etc. are used. Further, salts such as phosphates and magnesium salts are used as the inorganic salts. In addition, various organic substances and inorganic substances necessary for the growth of bacteria and enzyme production, or those containing them, such as yeast extract and vitamins, can be added to the medium. The culture is preferably liquid culture, and the 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 30 to 35 ° C under aerobic conditions, the culture time is about 12 hours to 3 days, and the amount of amylase produced is The culture may be terminated when the maximum is reached. The pH of the medium is preferably 8 or more, and about 9.5 is suitable for amylase production. The amylase of the present invention is mainly secreted and accumulated in the culture medium.

(Purification of enzyme) Purification of amylase from the obtained culture broth can be carried out according to a conventional method for obtaining an enzyme. First, a solid solution such as cells in the medium can be separated by an appropriate method such as filtration or centrifugation to obtain a supernatant. The crude product of amylase can be obtained by concentrating the separated solution and spray-drying it, freeze-drying it, salting out it, precipitating it with a hydrophilic organic solvent, and the like. For further purification, a method of using ion-exchange resin, a hydrophobic chromatography, a gel filtration method or the like alone or in combination can be used.

(Method for measuring enzyme titer) The enzyme titer of the amylase of the present invention can be measured by the following method. (1) 3,5-DNS (3,5-dinitrosalicylic acid)
Preparation of reagents 3,5-DN in 100 ml of 35% sodium hydroxide solution
Add 2.0 g of S and dissolve in a warm bath at 50-60 ° C. Then Rochelle salt (potassium sodium tartrate)
Add 20 g to dissolve and make up to 200 ml. (2) Activity measurement 100 mM glycine-N containing 1.0% soluble starch
0.1 ml of the enzyme solution is added to 0.4 ml of aCl-NaOH buffer (pH 10), and the mixture is reacted at 60 ° C for 10 minutes.
Immediately after completion of the reaction, water-cool the reaction mixture, and add 3,5-DNS reagent to 0.5
Add ml and boil for 5 minutes. After water cooling, deionized water 5.
Add 0 ml and color compare at 540 nm. Under the above conditions,
The amount of enzyme that produces a reducing sugar corresponding to 1 μmol of glucose per minute is 1 U.

(Enzyme Properties) Detailed properties of the amylase of the present invention will be described. (1) Action Both enzymes (amylase A, amylase B) act on starch or a partial degradation product thereof to hydrolyze α-1,4-glycoside bond, and mainly malto-oligosaccharide (G1
~ G4) is generated.

(2) Working pH and optimum pH By the same method as the above-mentioned activity measuring method, an acetate buffer solution (pH 4 to 4) is used.
6), Tris-HCl buffer (pH 7-8), glycine buffer (pH 9-11), Na ₂ HPO ₄ -NaOH
Soluble starch as substrate in buffer (pH 12) 6
When the activity is measured at 0 ° C., the optimum pH is around 10.5 for amylase A and around 10 for amylase B. Reaction p
The relationship between H and activity is shown in FIG. Both pH 4-12
Works with.

(3) Stable pH range Acetate buffer (pH 4-6), Tris-HCl buffer (pH 7-8.5), glycine buffer (pH 8.5-1)
1) or Na ₂ HPO ₄ -NaOH buffer (pH 1
After the enzyme solution prepared in 1) to 12) was left at 40 ° C. for 30 minutes, the residual activity was measured by the above activity measuring method. The stable pH is 6 to 11 for both amylase A and B. Treatment pH
The relationship between the residual activity and the residual activity is shown in FIG.

(4) Working temperature and optimum temperature When measured by the above-mentioned activity measuring method while changing the reaction temperature,
The optimum temperature for both amylase A and B is about 65 ° C. The relationship between reaction temperature and activity is shown in FIG. Both work over the entire measuring range (40 ° C to 80 ° C).

(5) Thermal stability Under a glycine buffer solution (pH 10), at various temperatures (40 ° C-
It was heat-treated at 90 ° C.) for 30 minutes, cooled with ice, and the residual activity was measured by the above-mentioned activity measuring method. When calcium was not added to both amylase A and B, the activity was decreased to about 60% at 50 ° C, but it was stable in the presence of ₂ mM CaCl ₂ even after the treatment at 60 ° C. The relationship between heat treatment temperature and residual activity is shown in FIG.

(6) Molecular Weight The molecular weights obtained by SDS-polyacrylamide gel electrophoresis are about 110,000 for amylase A and about 90,000 for amylase B.

(7) Degradation of Raw Starch Amylase A has a capability of degrading raw starch of potato and wheat.

Amylases A and B are strains of Bacillus sp. It is produced by strain SD773 and has an optimum pH in the alkaline region. As similar enzymes, there are alkali amylase described in JP-B-48-4553, alkali amylase described in JP-B-51-9033, heat-resistant alkali amylase described in JP-A-2-49584 and the like. Compared to the ones, the molecular weight is different, and it is clearly another enzyme.

(8) Effect of inhibitor The activity measured by the above-mentioned activity measuring method in the presence of various inhibitors is shown below, assuming that the activity in the absence of the inhibitor is 100%. Inhibitor Inhibitor concentration Active FeCl ₃ 1 mM 93% CuSO ₄ 〃 122% HgCl ₂ 〃 20% AgNO ₃ 〃 91% ZnSO ₄ 〃 90% EDTA 0.1 mM 7% SDS 0.1% 100% EDTA is ethylenediaminetetraacetic acid, SDS Is an abbreviation for sodium dodecyl sulfate.

[0026]

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

Example 1: Culture of amylase-producing bacterium 0.5% casamino acid, 2% starch (soluble), 0.3% yeast extract, 0.1% dipotassium hydrogen phosphate, magnesium sulfate heptahydrate 0 0.02%, sodium carbonate 0.35
The liquid medium consisting of 10% was sterilized by a conventional method and aseptically dispensed into a test tube and a Sakaguchi flask. The SD773 strain was inoculated into a test tube and shake-cultured at 33 ° C. for 24 hours to give a precultured cell suspension of which 0.1 ml was inoculated into the Sakaguchi flask containing the above-mentioned medium. After culturing at 33 ° C. for 21 hours, the culture solution was centrifuged, and the amylase activity of the supernatant was measured and found to be about 1.3 U / ml.

Example 2 Purification of Amylase The amylase of the present invention was purified from the supernatant obtained in Example 1. First, 3 from the supernatant by ammonium sulfate precipitation
A 0-85% saturated fraction precipitate was obtained. 20 mM of this precipitate
A DEAE (diethylaminoethyl) anion exchange resin column (16 mm) was dissolved in Tris-HCl buffer (pH 7.5), desalted by dialysis, and equilibrated with the same buffer.
Φ, 10 cm) and then a sodium chloride concentration gradient (0
-1M). Ammonium sulphate was added to the active fraction to a final concentration of 15% saturation and equilibrated with a buffer containing 15% saturated ammonium sulphate phenyl Toyopearl 650M (16 mmφ, 10 cm;
It was adsorbed on Tosoh Corporation (trade name). The active fraction eluted by applying a concentration gradient of 15-0% ammonium sulfate was concentrated by ultrafiltration,
Fractionation was performed using a Toyopearl HW-55 Superfine (trade name of Tosoh Corporation) column. When the active fraction was examined by polyacrylamide gel electrophoresis, two bands were found. It was confirmed by activity staining that each of the two bands had amylase activity. Further, after cutting out an active band from this gel, each enzyme was extracted with 20 mM Tris-HCl buffer (pH 7.5) (amylase A, B). When the activities of these enzyme solutions were measured by the above-mentioned activity measuring method, they were purified 237 times and 236 times, respectively.

[0029]

INDUSTRIAL APPLICABILITY The present enzyme is an enzyme which exhibits high activity in high temperature and alkaline regions. At present, amylases that act at high temperatures are all neutral enzymes, and alkaline enzymes have the drawbacks that they are sensitive to heat. Enzymes that satisfy both of them are not yet on the market. By using this enzyme, starch decomposition in a high temperature alkaline region can be significantly improved. For example, in the textile industry, it can be used in a desizing step under alkaline conditions during a scouring and bleaching step, and a texture different from the conventional one can be obtained. In addition, since it has high stability in alkali, it can be used as a detergent enzyme and can enhance the detergency. Furthermore, by adding it to an automatic dishwashing detergent, 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. In addition, the Bacillus sp. By culturing the SD773 strain,
The enzyme of the present invention can be efficiently obtained.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between reaction pH and activity of amylase of the present invention.

FIG. 2 is a graph showing the treatment pH and residual activity of the amylase of the present invention.

FIG. 3 is a graph showing the relationship between reaction temperature and activity of amylase of the present invention.

FIG. 4 is a graph showing the relationship between the heat treatment temperature and the residual activity of the amylase of the present invention.

Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location (C12N 1/20 C12R 1:07) (C12N 1/21 C12R 1:07)

Claims (7)

[Claims] 1. An amylase having the following properties. (1) Action It acts on starch to hydrolyze the α-1,4-glycoside bond. (2) Optimum pH When a soluble starch is used as a substrate and reacted at 60 ° C. for 10 minutes, the optimum pH is about 10.5. (3) Optimum temperature When soluble starch is used as a substrate and reacted at pH 10 for 10 minutes, the optimum temperature is about 65 ° C. (4) Stable pH When treated at 40 ° C for 30 minutes, it is stable in the pH range of 6-11. (5) Thermal stability When treated at pH 10 for 30 minutes, it has a residual activity of 100% up to 60 ° C in the presence of 2 mM calcium chloride, and a residual activity of about 60% at 50 ° C in the absence thereof. (6) Molecular weight The molecular weight measured by SDS-polyacrylamide gel electrophoresis is about 110,000. 2. An amylase having the following properties. (1) Action It acts on starch to hydrolyze the α-1,4-glycoside bond. (2) Optimum pH When a soluble starch is used as a substrate and reacted at 60 ° C. for 10 minutes, the optimum pH is about 10. (3) Optimum temperature When soluble starch is used as a substrate and reacted at pH 10 for 10 minutes, the optimum temperature is about 65 ° C. (4) Stable pH When treated at 40 ° C for 30 minutes, it is stable in the pH range of 6-11. (5) Thermal stability When treated at pH 10 for 30 minutes, it has a residual activity of about 90% at 60 ° C in the presence of 2 mM calcium chloride, and a residual activity of about 60% at 50 ° C in the absence. (6) Molecular weight The molecular weight measured by SDS-polyacrylamide gel electrophoresis is about 90,000. 3. A method for producing amylase, which comprises culturing a bacterium belonging to the genus Bacillus having the ability to produce amylase according to claim 1 and collecting the amylase from the culture. 4. A method for producing amylase, which comprises culturing a bacterium of the genus Bacillus having the ability to produce amylase according to claim 2 and collecting the amylase from the culture. 5. A bacterium of the genus Bacillus is Bacillus sp. SD7
The method for producing amylase according to claim 3 or 4, which is strain 73 (FERMP-14462) or a mutant strain or genetically engineered strain thereof. 6. A bacterium belonging to the genus Bacillus having the ability to produce the amylase according to claim 1. 7. Bacillus sp. SD773 strain (FERM
P-14462) or a mutant strain or a genetically engineered strain thereof.