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Method of improving alpha-amylase activity in bacillus subtilis fermentation liquid

A technology of Bacillus subtilis and amylase is applied in the field of improving α-amylase activity in Bacillus subtilis fermentation broth, and can solve problems such as no reported results yet

Active Publication Date: 2017-05-31
河南崤函生物科技有限公司
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Problems solved by technology

[0004] Based on the above research, if the sdpC gene in Bacillus subtilis can be mutated to reduce the formation of Sdp, it is possible to greatly improve the fermentation and cultivation effect of Bacillus subtilis, but in the prior art, no good reports have been seen yet achievement

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  • Method of improving alpha-amylase activity in bacillus subtilis fermentation liquid
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  • Method of improving alpha-amylase activity in bacillus subtilis fermentation liquid

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specific Embodiment

[0048] In the following, the present application will be further described in conjunction with the examples. Before introducing the specific examples, a brief introduction of some experimental backgrounds in the following examples will be given as follows.

[0049] biomaterials:

[0050]Bacillus subtilis 168 strain and Bacillus subtilis integrated plasmid pMUTIN4 were purchased from Hangzhou Baosai Biotechnology Co., Ltd.;

[0051] Primer sequence synthesis and sequencing were provided by Beijing Huada Gene Company;

[0052] Experimental reagents:

[0053] LB liquid medium: tryptone 10g, yeast extract 5g, sodium chloride 10g, use deionized water to make up to 1000mL, heat moderately to make it fully dissolved, divide into 500mL Erlenmeyer flasks according to the amount of 100mL per bottle, 121 Sterilize at ℃ for 30min; (LB solid medium, add 20g agar powder in addition);

[0054] Kit for PCR amplification, product of Beijing Kangrun Chengye Biotechnology Co., Ltd.;

[0055]...

Embodiment 1

[0059] Since the construction of the recombinant plasmid based on the plasmid pMUTIN4 is the basis of this application, the specific process is as follows.

[0060] (1) Design primers, PCR clone and amplify sdpBC partial sequence DNA

[0061] First, the primer sequences for PCR amplification were designed as follows:

[0062] sdp F: 5'-CGGAATTCTAAAGAGTGGGCAGAAGGAAC-3', (the GAATTC partial sequence at the 5' end is the EcorI restriction site)

[0063] sdp R: 5'-CGGGATCCTTTACTAACTTTTTTACCTTCTGT-3'; (the 5' GGATCC partial sequence is the BamHI restriction site)

[0064] Secondly, use the Bacillus subtilis 168 genomic DNA (DNA extraction is carried out using a cell / bacteria / yeast genomic DNA mini-extraction kit, which was purchased from Shanghai Laifeng Biotechnology Co., Ltd.) as a template for PCR amplification. The system design is as follows:

[0065] Bacillus subtilis 168 genomic DNA, 1 μL;

[0066] Primer sdp F, 1 μL;

[0067] Primer sdp R, 1 μL;

[0068] GenStar 2×Taq...

Embodiment 2

[0100] The recombinant plasmid constructed in Example 1 was transformed into Bacillus subtilis 168, and further screened to obtain the recombined mutant strain, the technical principle of its genome transformation of Bacillus subtilis 168 is as follows figure 1 As shown, genome integration is performed based on the principle of homologous single crossover, and the specific transformation and screening process are briefly introduced as follows.

[0101] The homologous single exchange recombinant plasmid constructed in Example 1 was transformed into Bacillus subtilis 168, and detected and identified

[0102] The homologous single-crossover recombinant plasmid constructed in Example 1 was transformed into Bacillus subtilis 168 by Spizizen low-salt environment competent transformation method, and the specific process was as follows.

[0103] (1) Preparation of competent cells of Bacillus subtilis 168 strain

[0104] The Bacillus subtilis 168 strain stored in a -80°C ultra-low te...

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Abstract

The invention belongs to the technical field of biological fermentation engineering and particularly relates to a method of improving alpha-amylase activity in bacillus subtilis fermentation liquid. A bacillus subtilis strain genome is modified by a gene engineering method, and the method particularly comprises the steps of PCR clonal expansion of sdpBC partial sequence DNA, enzyme digestion and connection, conversion, screening, identification, conversion from homologous single exchange recombinant plasmid to bacillus subtilis 168, detection and identification. According to the method, a bacillus subtilis 168 strain having a relatively clear physiology and conversion background and completing sequencing is taken as a research example, and a sporulation delay protein gene sdpC is subjected to insertion mutation by a homologous recombination method. A preliminary study shows that the viable count and the alpha-amylase activity of the modified strain obtained by the method are better increased and improved, the strain has a better application value, and an application foundation is laid for modification of other strains.

Description

technical field [0001] The application belongs to the technical field of biological fermentation engineering, and in particular relates to a method for improving the activity of α-amylase in Bacillus subtilis fermentation broth. Background technique [0002] Bacillus subtilis is a kind of widely distributed Gram-positive rod-shaped aerobic bacteria, non-pathogenic, not easy to produce drug resistance, good compatibility with the environment, and has a strong ability to secrete various enzymes and antibiotics outside the cell . Bacillus subtilis is widely used, and currently involves various fields such as fermentation, pest control, aquaculture, and sewage treatment. However, in the initial stage of spore formation, Bacillus subtilis delays spore formation through the process of "cannibalism", that is, the cells that form spores produce and release bacterial toxins Skf (spore formation suicide factor) and Sdp ( spore formation delay protein), at the same time, it has an im...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N9/28C12R1/125
CPCC07K14/32C12N9/2417C12Y302/01001
Inventor 焦国宝孙利鹏邱立友高玉千黄涛田芳刘仲敏
Owner 河南崤函生物科技有限公司
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