Bacillus subtilis yielding mesothermal alpha-amylase

A Bacillus subtilis and amylase technology, applied in the field of microorganisms, can solve the problems of consumption of reagents, increase of technological process, poor enzyme stability, etc., and achieve the effects of wide pH value range, high enzyme production activity, and high enzyme activity.

Active Publication Date: 2015-03-25
湖南新鸿鹰生物工程有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the poor stability of this type of enzyme, and when the pH is lower than 5.0, it will be seriously inactivated, which limits its application and brings certain troubles to the industrial production process.
For example, in the saccharification and liquefaction process of starchy raw materials, since the pH of starch slurry is generally around 4.5, and the applicable pH of widely used α-amylase is 6.0-7.0, it is necessary to adjust the pH of starch slurry at t

Method used

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  • Bacillus subtilis yielding mesothermal alpha-amylase

Examples

Experimental program
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Example Embodiment

[0024] Example 1: UV-Lithium Chloride-Diethyl Sulfate Compound Mutagenesis Screening

[0025] (1) Preparation of bacterial suspension

[0026] The single colony of the starting strain grown on the plate was added to the seed culture medium and cultured at 100 r / min at 37°C for 24 hours. After centrifugation, 1 mL of the culture broth was centrifuged and washed twice with normal saline, and resuspended in 9 mL of normal saline. in.

[0027] (2) UV-Lithium Chloride-Diethyl Sulfate Compound Mutagenesis

[0028] Place 3 mL of bacterial suspension in a sterile plate, stir and irradiate it under a UV lamp with a distance of 30 cm and a power of 15 W for 60 seconds. After the irradiated bacterial liquid is serially diluted (10 -1 -10 -5 ) Spread on a lithium chloride plate, and dilute the plate with bacterial solution without ultraviolet irradiation as a control. Wrap the above-mentioned uniformly coated plate with black cloth or newspaper, and incubate at 37°C for 48 hours. On the plate w...

Example Embodiment

[0037] Example 2: Optimization of fermentation conditions

[0038] A preparation method of medium temperature α-amylase includes the following steps:

[0039] (1) Strain activation

[0040] Inoculate the well-preserved slant strain of Bacillus subtilis 304 on the slant medium, culture at 35°C for 30h to activate the strain, and activate it 3 times;

[0041] The slant culture medium consists of 6 g beef extract, 8 g sodium chloride, 15 g peptone, 5 g glucose, 20 g agar, 1000 mL distilled water, pH 6.5, and sterilization at 121°C for 20 min;

[0042] (2) Expanded cultivation of liquid seeds

[0043] ①First-level seed culture: Connect the 2 loops of slant strains after activation in step (1) into a 500ml shaker flask, fill the medium with 100ml, rotate the shaker at 250 rpm, culture temperature 35℃, culture time 10h;

[0044] ②Secondary seed culture: Put the first-level seed into a 500ml second-level seed shaker at 10% of the inoculum, and the cultivation conditions are the same as those of...

Example Embodiment

[0061] Example 3: Determination of amylase activity

[0062] (1) Definition of enzyme activity unit: 1 mL of crude enzyme solution, liquefying 1 mg of soluble starch in 1 min at 65°C and pH 5.0, is 1 unit of enzyme activity, expressed in U / mL.

[0063] (2) Use DNS method to measure amylase activity.

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Abstract

The invention discloses bacillus subtilis yielding mesothermal alpha-amylase. The strain is obtained by compound mutation by virtue of lithium chloride-diethyl sulfate. The bacillus subtilis 304 is specifically Bacillus subtilis 304, wherein the preservation number is CCTCC NO: M 2013600. The optimum action temperature of the mesothermal alpha-amylase yielded by bacillus subtilis is 65 DEG C and the mesothermal alpha-amylase which is preserved at 65 DEG C for 24 hours still has 80% enzyme activity and which is preserved at 70 DEG C for 12 hours still has 50% enzyme activity; the optimum reaction pH value of the enzyme is 5.0 and the mesothermal alpha-amylase which is preserved at pH of 4-6 for 18 hours still has over 80% enzyme activity; the enzyme activity is 7000-8500U/ml. The bacillus subtilis is particularly suitable for industrial demand, wherein the reaction temperature is high and a liquefaction process and a saccharification process exist.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Bacillus subtilis producing mesophilic α-amylase. Background technique [0002] The full name of α-amylase is α-1,4-glucan hydrolase (EC3.2.1.1). When acting 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. According to the temperature range of its use, it is divided into high temperature α-amylase, medium temperature α-amylase and α-amylase. In different industries, it is necessary to selectively use α-amylases with different enzymatic properties. [0003] Medium-temperature α-amylase is a kind of amylase that is used at a temperature of 60-90°C, and is relatively stable at pH 6.0-7.0 below 60°C. Widely used in the liquefaction of alcohol, beer, monosodium...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/28C12R1/125
CPCC12N9/2417C12Y302/01001C12N1/205C12R2001/125
Inventor 李洪兵张锦杰李海清胡永明朱永明孙明芳
Owner 湖南新鸿鹰生物工程有限公司
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