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A kind of recombinant Beauveria bassiana protease k and its industrial production and purification method

A Beauveria bassiana and protease technology, applied in the fields of genetic modification and protein engineering, can solve problems such as reports on industrial production of Beauveria bassiana recombinant proteinase K, which are not found, achieve comprehensive recovery rate improvement, reduce use costs, Overcome the effects of low yield

Active Publication Date: 2018-06-08
合肥巅峰生物科技有限公司
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Problems solved by technology

So far, no reports on the industrial production of Beauveria bassiana recombinant proteinase K have been found in patent documents and non-patent documents

Method used

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  • A kind of recombinant Beauveria bassiana protease k and its industrial production and purification method
  • A kind of recombinant Beauveria bassiana protease k and its industrial production and purification method
  • A kind of recombinant Beauveria bassiana protease k and its industrial production and purification method

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Embodiment Construction

[0039] In order to make the technical means, creative features, goals and effects achieved by the present invention easy to understand, the present invention will be further described below in conjunction with specific embodiments.

[0040] 1) Acquisition of Beauveria bassiana proteinase K Pichia pastoris recombinant expression optimization coding gene:

[0041]The original cDNA sequence of Beauveria bassiana protease K (Gene ID: AY520815, SEQ ID NO.2) was obtained from the GeneBank database. The rare triplet codons in the gene were replaced with the corresponding codon nucleotides with the highest abundance of tRNA in Pichia pastoris cells, and finally the optimized gene coding sequence of Beauveria bassiana proteinase K suitable for recombinant expression of Pichia pastoris was obtained. During the optimization process, the signal peptide coding segment in the original cDNA sequence of Beauveria bassiana proteinase K was removed, and the final number of A+T bases reached 50%...

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Abstract

The invention discloses a recombinant beauveria brongniartii proteinase K as well as an industrial production and purification method thereof and relates to the technical field of gene modification and protein engineering. Recombinant beauveria brongniartii proteinase K coding genes suitable for the pichia pastoris recombinant expression are obtained with a biological information means, optimization genes are synthesized and successfully expressed in a eukaryotic system, and the recombinant proteinase K with the enzymatic activity obviously higher than that of a traditional tritirachium album limber proteinase K. According to the recombinant beauveria brongniartii proteinase K as well as the industrial production and purification method thereof, the coding genes are synthesized chemically, and GS115 / pPIC9K-ProK high-yield engineering bacteria are established, so that defects of the low yield and the high cost of natural products are overcome, the usage cost of the proteinase K in related industries is significantly reduced, and the recombinant beauveria brongniartii proteinase K as well as the industrial production and purification method thereof have broad application prospects.

Description

Technical field: [0001] The invention relates to the technical fields of gene modification and protein engineering, in particular to a recombinant Beauveria bassiana protease K and its industrial production and purification method. Background technique: [0002] Proteinase K (Proteinase K, EC 3.4.21.64, also known as endopeptidase K, saprophytic fungal alkaline protease, Candida albicans serine protease, Candida albicans protease K), is a serine protease with broad cutting activity. The protein degrades natural keratin (keratin) by cleaving the carboxy-terminal peptide bonds of aliphatic and aromatic amino acids, hence its name. Because proteinase K can exist stably in guanidine hydrochloride (3M), urea (4M) and SDS (0.5-1%) and has the ability to degrade proteins, it is widely used in the preparation of chromosomal DNA for pulse electrophoresis, Western blot and removal Nucleases in DNA and RNA preparations. [0003] Proteinase K was first purified from Candida albicans L...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/58C12N15/57C12N15/81
CPCC12N9/58C12Y304/21064
Inventor 丁海平李坤梁良
Owner 合肥巅峰生物科技有限公司
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