A Streptomyces trypsin gm2938 and its heterologous expression in Bacillus subtilis

A technology of GM2938 and Bacillus subtilis is applied in the field of heterologous expression of Streptomyces trypsin GM2938, which can solve the problems of inability to meet industrial requirements, low enzyme yield, and difficult control of industrial fermentation.

Active Publication Date: 2021-11-05
SICHUAN UNIV
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Problems solved by technology

[0003] Compared with trypsin derived from mammals, microbial fermentation production can obtain trypsin with a single component and similar applications. However, due to the long fermentation period of Streptomyces itself, the industrial fermentation is not easy to control, the yield is low, and the secondary metabolites in the later stage affect the subsequent production. Due to the shortcomings of enzyme purification and other shortcomings, researchers expect to use genetic engineering to obtain heterologous expression of Streptomyces trypsin
At present, the research on Streptomyces trypsin still has the following problems: (1) The development of trypsin-producing Streptomyces is insufficient, and there are few types of Streptomyces trypsin; (2) The content of GC in the Streptomyces trypsin gene is high, and it is difficult to achieve active Heterologous expression; (3) Enzyme yield is low, unable to meet industrial requirements
[0004] With the in-depth study of Streptomyces trypsin-encoding genes, some Streptomyces trypsins have been expressed in heterologous hosts; among them, Streptomyces freundii tryptase SFT is in E. coli The system is expressed in the form of zymogen, in Pichia The system is expressed in the form of a mature enzyme; the heterologous expression of Streptomyces griseus trypsin SGT has been studied more, and heterologous expression has been achieved in Escherichia coli, Streptomyces, Bacillus subtilis and Pichia pastoris respectively, but due to The GC content in the Streptomyces trypsin coding gene is high, and the protein itself contains three pairs of disulfide bonds, which makes its expression efficiency and yield low in most expression systems; therefore, choose a suitable signal peptide to construct a recombinant expression plasmid , which can provide guidance for the subsequent development and heterologous expression of other Streptomyces trypsins

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  • A Streptomyces trypsin gm2938 and its heterologous expression in Bacillus subtilis
  • A Streptomyces trypsin gm2938 and its heterologous expression in Bacillus subtilis
  • A Streptomyces trypsin gm2938 and its heterologous expression in Bacillus subtilis

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Embodiment 1

[0013] Embodiment one: the acquisition of trypsin GM2938 gene

[0014] Genomic DNA of Streptomyces endophytic strain A249 was extracted, and the whole genome sequence was sequenced (completed by Beijing Novogene Biotech), combined with the obtained ORF reading frame, after reviewing relevant literature, comparing databases, and analyzing feasibility studies , A gene that is better than trypsin GM2938 was selected, and the amino acid sequence encoded by it was 80.85% similar to the known trypsin SGT from Streptomyces griseus.

Embodiment 2

[0015] Example 2: Heterologous expression of GM2938 mature peptide gene

[0016] Design specific primers to amplify the mature peptide gene of GM2938, upstream primer (5'-AGGAGGCGCAACTCAAGCTTTTGCAGTCGTCGGCGGCACGCGCGCCG-3'), downstream primer (5'-AGCTTGCATGCCTGCAGGTCGACTCTCAGAGACCGGCGG CCGCTCTGGCT-3'), design plasmid pWB980 linearization primer, upstream primer (5'- AGCCAGAGCGGCCGCCGGTCTCTGAGAGTCGACCTGCAGGCATGCAAGCT-3'), downstream primers (5'-CGGCGCGCGTGCCGCCGACGACTGCAAAAGCTTGAGT TGCGCCTCCT-3'), obtained the amplified mature peptide gene of GM2938 and the linearized plasmid pWB980 fragment by PCR respectively, purified the recovered linearized plasmid and the target The gene fragments serve as primers for each other, and POE-PCR is carried out by ultra-high-fidelity enzymes, and the obtained plasmid polymer is transformed into the Bacillus subtilis engineering bacterium SCK6 to obtain the genetic engineering expression bacterium.

Embodiment 3

[0017] Embodiment three: Recombinant Bacillus subtilis engineered bacterium culture condition

[0018] Engineering bacteria containing recombinant plasmid pWB980-mt2938 B. subtilis SCK6 was inoculated in 100 mL LB-resistant medium containing 1 µg / mL erythromycin and 50 µg / mL kanamycin, cultured on a shaker at 37°C overnight, and the next day, the above seed solution was inoculated at 2% inoculum In the fermentation medium with the same resistance (medium composition: 15g / L soluble starch, 16 g / L tryptone, 32 g / L soybean peptone, 72 mM K 2 HPO 4 , 17 mM KH 2 PO 4 ; the initial pH of the medium was 8.0), 34 °C, 200 rpm shaker culture for 72 h; since the promoters carried by the plasmid pWB980 are all constitutive promoters, no additional inducer is needed to induce protein expression, and the target gene A signal peptide was added to promote the extracellular expression of the protein, so it was necessary to collect the supernatant obtained after centrifugation and measure...

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Abstract

The invention discloses a streptomyces trypsin gene, construction of a heterologous expression carrier and enzymatic properties of the enzyme, belonging to the field of biotechnology. The full length of the GM2938 gene described in the present invention is obtained by mining the full gene data of Streptomyces endogenous A249. The full length of the gene is 759 bp, encoding 253 amino acids, and the gene is expanded in vitro by PCR. The present invention realizes the heterologous cloning, expression and purification of trypsin GM2938 gene in Bacillus subtilis SCK6 for the first time, and carries out the characterization of trypsin properties. The trypsin has good enzymatic properties, which will have broad prospects in the application of leather technology and food industry.

Description

technical field [0001] The invention relates to the technical field of heterologous expression of streptomyces trypsin GM2938 and its application. Background technique [0002] Streptomyces trypsin is an important member of the microbial trypsin family because of its good biological safety, controllable production quality, non-calcium ion activation, homology with mammalian bovine trypsin and enzymatic properties It is also similar to bovine trypsin and other advantages, and is widely used in pharmaceuticals, clinical diagnosis, food processing, leather processing, biochemical detection and other fields. [0003] Compared with trypsin derived from mammals, microbial fermentation production can obtain trypsin with a single component and similar applications. However, due to the long fermentation period of Streptomyces itself, the industrial fermentation is not easy to control, the yield is low, and the secondary metabolites in the later stage affect the subsequent production....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/76C12N15/75
CPCC12N9/6427C12N15/75C12Y304/21004
Inventor 田永强王志宽龙秀锋
Owner SICHUAN UNIV
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