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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 this stage to adapt to starch. The liquefaction process is under the action conditions of α-amylase, and in the subsequent saccharification process, the pH of the action of glucoamylase is generally 4.2-5.0. In order to apply the action conditions of glucoamylase, the pH of the system needs to be adjusted here. Due to the repeated pH Adjustment, not only increases the process, consumes reagents, but also introduces foreign ions, which increases the burden of classification

Method used

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  • Bacillus subtilis yielding mesothermal alpha-amylase

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Effect test

Embodiment 1

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

[0025] (1) Preparation of bacterial suspension

[0026] Inoculate the single colony of the starting strain grown after streaking on the plate into the seed medium, culture at 37°C for 24 hours at 100 r / min, take 1 mL of the culture medium, centrifuge, wash twice with normal saline, and resuspend with 9 mL of normal saline middle.

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

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

Embodiment 2

[0037] Embodiment 2: optimization of fermentation conditions

[0038] A preparation method for mesophilic α-amylase, comprising the steps of:

[0039] (1) Activation of bacteria

[0040] The well-preserved slant strain of Bacillus subtilis 304 was inoculated on the slant medium, cultured at 35°C for 30 hours to activate the strain, and activated 3 times in this way;

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

[0042] (2) Expanded cultivation of liquid seeds

[0043] ①First-level seed culture: Put the slant bacteria activated in step (1) into a 500 ml shake flask with 2 loops, the medium capacity is 100 ml, the rotary shaker is 250 rpm, the culture temperature is 35°C, and the culture time is 10h;

[0044] ②Cultivation of secondary seeds: Inoculate 10% of the primary seeds into 500 ml secondary seed shake flasks, and the cultivation condit...

Embodiment 3

[0061] Embodiment 3: the mensuration of amylase activity

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

[0063] (2) The amylase activity was determined by DNS method.

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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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