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Bacillus subtilis producing high-temperature-resistance alpha-amylase

A Bacillus subtilis, high temperature-resistant technology, applied in the field of microorganisms, can solve the problems of complex post-processing procedures, low enzyme activity, and high production costs, and achieves a wide range of optimal pH values ​​for enzyme action, high enzyme production activity, and high enzyme activity. Effect

Inactive Publication Date: 2015-05-20
邵素英
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, although many skilled in the art have carried out tireless research on the production of high-temperature-resistant α-amylase, there are still the following deficiencies: 1. low enzyme activity, complicated post-treatment procedures, and relatively high production costs; 2. tolerance The temperature is difficult to exceed 95°C, and the liquefaction effect is not ideal; 3. The optimum pH range is narrow, which is not suitable for the "two-enzyme method" production process; 4. The fermentation and extraction process contains impurities, and the application range is limited

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: UV-Lithium Chloride compound mutagenesis screening

[0038] (1) Preparation of bacterial suspension

[0039] Inoculate the single colony of Li-2010 grown after streaking on the plate into the seed medium, culture at 40°C for 12 hours at 100 r / min, take 1 mL of culture medium, centrifuge, wash twice with normal saline, and resuspend with 9 mL of physiological in salt water.

[0040] (2) Ultraviolet-Lithium Chloride-Diethyl Sulfate Compound Mutagenesis

[0041] Place 3mL of bacterial suspension on a sterile plate, stir and irradiate for 100s under a UV lamp with a distance of 30cm and a power of 15w. Spread the irradiated bacterial solution on the lithium chloride plate after gradient dilution (10-1-10-5), and use the diluted bacterial solution without UV irradiation as a control. Wrap the uniformly coated plate above with black cloth or newspaper, incubate at 40°C for 48 hours, screen out the plate with the largest ratio of hydrolysis circle to colony diame...

Embodiment 2

[0050] Embodiment 2: the mensuration of amylase activity

[0051] (1) Definition of enzyme activity unit: 1 mL of crude enzyme solution, at 110°C and pH 4.5, liquefies 1 mg of soluble starch in 1 minute, which is 1 enzyme activity unit, expressed in U / mL.

[0052] (2) The amylase activity of Li-2010, Li-2013-A, Li-2013-02, and Li-2013-B was measured by DNS method. It was known that Li-2013-02 had the highest stable enzyme activity, reaching 31500U / mL.

Embodiment 3

[0053] Example 3: Determination of enzymatic properties Determination of enzymatic properties

[0054] (1) Effect of temperature on enzyme activity and enzyme thermal stability

[0055] In the same reaction system, the enzyme activity of the amylase was measured at 80-110°C, and the results showed that the enzyme showed high enzymatic activity between 90-110°C, indicating that the temperature adaptability range of the enzyme is wide, and its optimum effect The temperature is 100-110°C.

[0056] (2) Effect of pH on enzyme activity and acid stability

[0057] In the same reaction system, the enzyme activity of the enzyme was measured at pH 3-8.5, and the results showed that the enzyme activity was higher at pH 3-7, indicating that the enzyme was acid amylase. Stability of the enzyme at different pH values: Dilute the enzyme with disodium hydrogen phosphate-citric acid buffer with different pH values, and measure the enzyme activity after incubation at 25°C for 18 hours. The re...

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PUM

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Abstract

The invention discloses bacillus subtilis producing high-temperature-resistance alpha-amylase. The bacillus subtilis Li-2013-02 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on July 15, 2013, and has a preservation number of CGMCC No.7926. The strain is prepared from bacillus subtilis Li-2010 producing high-temperature-resistance alpha-amylase by means of ultraviolet ray-lithium chloride-diethyl sulfate combined mutation screening, has the characteristics of strong acid resistance, heat resistance and high enzyme production activity, and has a wide application prospect.

Description

technical field [0001] The invention belongs to the technical field of microbes, and in particular relates to a strain of bacillus subtilis producing high-temperature resistant alpha-amylase. Background technique [0002] The full name of α-amylase is α-1,4-glucan hydrolase (EC3.2.1.1). When it acts on starch, it can cut α-1,4-glucosidic bonds from the inside of the molecule to generate dextrin and reduce Sugar, because the C1 carbon atom of the terminal glucose residue of the product is in the α-configuration, so it is named α-amylase. [0003] Thermostable α-amylase widely exists in animals (saliva, pancreas), plants (barley) and microorganisms, and is an endoglucosidase. It can rapidly hydrolyze the α-1,4 glucosidic bond in the starch molecule at a higher temperature, arbitrarily cut the starch into short-chain dextrins of different lengths and a small amount of oligosaccharides, glucose and maltose, thereby making the starch paste Its viscosity drops rapidly, and it is...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N15/01C12N13/00C12R1/125
CPCC12N1/20C12N13/00C12N15/01C12N1/205C12R2001/125
Inventor 李绩苏俊王玉李政
Owner 邵素英
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