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A strain of Bacillus subtilis with high production of mesophilic α-amylase and its liquid fermentation method

A technology of Bacillus subtilis and amylase, applied in the field of microbial breeding and fermentation, can solve the problem of unsatisfactory maximum enzyme activity, and achieve the effects of wide pH value range, high fermentation enzyme activity and strong antibacterial performance

Active Publication Date: 2019-05-14
SHANDONG LONGKETE ENZYME PREPARATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the maximum enzyme activity of the existing mesophilic α-amylases during fermentation is still unsatisfactory, so there is an urgent need to develop strains and fermentation methods of mesophilic α-amylases with higher yields and shorter fermentation cycles to meet the increasing Growing demand for industrialized production

Method used

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  • A strain of Bacillus subtilis with high production of mesophilic α-amylase and its liquid fermentation method
  • A strain of Bacillus subtilis with high production of mesophilic α-amylase and its liquid fermentation method
  • A strain of Bacillus subtilis with high production of mesophilic α-amylase and its liquid fermentation method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1: the breeding of bacterial strain

[0023] (1) Bacterial suspension preparation:

[0024] Centrifuge the Bacillus subtilis BS2007 bacterial solution cultivated to the logarithmic phase at 8000 rpm for 5 minutes, discard the supernatant, and resuspend it with 10% glycerol to obtain the bacterial suspension. The cell concentration of the bacterial suspension is at 10 7 pieces / ml.

[0025] (2) ARTP mutagenesis:

[0026] Suck 10 μL of bacterial suspension into the center of the slide and spread it evenly; use sterile tweezers to place the slide on the corresponding hole on the rotating platform of the ARTP system operating room, adjust the knob below the slide to make the slide 2mm from the airflow port, and set The air flow is 10SLM, the power is adjusted to 100W, and the mutagenic irradiation time is selected as 20s, 25s, and 30s respectively; after the sample is processed, use sterile tweezers to move the slide to the EP tube containing 1mL of normal salin...

Embodiment 2

[0033] Embodiment 2: Liquid fermentation produces mesophilic alpha-amylase

[0034] The liquid fermentation method after the optimization of the medium formula and the optimization of the fermentation process is as follows.

[0035] Technological process: preservation of strains→slope activation→plate culture→seed liquid shake bottle culture→fermentation culture in 50L fermenter→extraction and refining

[0036] Seed culture: The 3 mutagenic strains screened by shake flasks were cultured in conventional shake flasks to make seed liquid, and the volume of liquid in a 500mL Erlenmeyer flask was 50mL. The shake flask medium formula is: 3% soluble starch, 0.3% yeast extract, 0.3% peptone, 0.2% calcium chloride, the rest is water, pH 6.5; the culture conditions are: temperature 37°C, rotation speed 200rpm, culture period 6h;

[0037] Liquid fermentation: Liquid fermentation is carried out in a 50L fermenter. The composition of the fermentation medium is: 6% corn flour, 3% bean cake ...

Embodiment 3

[0046] Embodiment 3: the antibacterial property test of mutant strain

[0047] Preparation of the crude cell extract: take the fermentation liquid of the strain BS-122 fermented to 40h, centrifuge at 8000r / min at 4°C for 5min to remove the cell, take the supernatant and filter it through a bacterial filter (0.22μm) to obtain the crude cell extract .

[0048] Use the Oxford cup quantitative diffusion method to test the resistance to the test fungus: put the sterilized Oxford cup into the center of a petri dish with a diameter of 9 cm, pour the melted solid PDA medium into the plate under aseptic conditions, and wait for it to solidify Finally, take out the Oxford cup, inoculate 3 pieces (diameter 5mm) of indicator bacteria of substantially the same size on the edge of the petri dish equidistantly, and add 180 μl of the crude extract of the mutant bacteria or the original bacteria to the Oxford cup in the medium. Cultivate at 28°C, and measure the diameter of the inhibition zon...

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Abstract

The invention provides bacillus subtilis mutant strain of high-yield mesophilic alpha-amylase and a liquid fermentation method for the bacillus subtilis mutant strain. The strain is obtained through atmospheric and room temperature plasma (ARTP) mutation breeding, and the collection number of the strain is CGMCC No.10786. By fermentation condition optimization, average fermentation enzyme activity can reach 7309mu / mL, and the maximum average fermentation enzyme activity is 7400 mu / mL; compared with an original strain before mutation, the strain has a better antibacterial effect; the optimum temperature of the mesophilic alpha-amylase produced by liquid fermentation of the strain is 60 to 70 DEG C, and after being stored for 12h at 60 to 70 DEG C, the enzyme still has more than 75 percent of enzyme activity; the optimum reaction pH of the enzyme is 5.2 to 6.2, and after being stored for 12h at pH 5.2 to 6.2, the enzyme still has more than 85 percent of enzyme activity.

Description

technical field [0001] The invention relates to a Bacillus subtilis strain with high yield of mesophilic α-amylase and a liquid fermentation method thereof, belonging to the technical field of microbial breeding and fermentation. Background technique [0002] The full name of α-amylase is α-1,4-glucan hydrolase (EC 3.2.1.1). When it acts on starch, it can cut α-1,4-glucosidic bonds from the inside of the molecule to generate dextrin and reducing sugar , Because the C1 carbon atom of the terminal glucose residue of the product is in the α-configuration, it is named α-amylase. Amylase is the earliest to achieve industrial production and is by far the most widely used and the largest yield of enzyme preparations. [0003] Among them, mesophilic α-amylase is a kind of α-amylase whose optimum reaction temperature is 50-70°C. At present, mesophilic α-amylase has been widely used in modified starch and starch sugar, baking industry, beer brewing, alcohol industry and other indust...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/28C12R1/125
Inventor 王兴吉郭庆文高晓娜张杰
Owner SHANDONG LONGKETE ENZYME PREPARATION
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