Protease K multi-copy strain construction method

Abstract

The invention discloses a protease K multi-copy strain construction method, and belongs to the technical field of biology. According to the protease K multi-copy strain construction method, protease Kmulti-copy strains is artificially constructed in vitro by utilizing a Biobrick method, and the protease K multi-copy strain construction method mainly comprises the following specific steps: S1, constructing a multi-copy vector; S2, screening multi-copy strains; and S3, carrying out multi-copy strain large-scale fermentation. A protease K multi-copy tandem expression cassette is artificially constructed in vitro by using a Biobrick method; the construction method has the advantages that the copy number of recombinant plasmids is controllable, dependence on screening of high-concentration resistant drugs is not needed, large-scale screening is also not needed, the gene dose effect is considered as the most important factor for expression of foreign protein by pichia pastoris, and the expression quantity of the multi-copy strains is many times higher than that of single-copy strains; therefore, the yield of the protease K is increased, the price is lower, and the protease K can be applied to various fields.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing proteinase K multi-copy strains. Background technique [0002] Proteinase K belongs to serine proteases, which is the main protease produced by Candida albicans Limberella; because the microorganisms that can synthesize this kind of protease can grow in the environment with keratin as the only carbon and nitrogen source, it is called Proteinase K; proteinase K has extremely high enzyme activity and broad substrate specificity, and can preferentially decompose ester bonds and peptide bonds adjacent to the C-terminus of hydrophobic amino acids, sulfur-containing amino acids, and aromatic amino acids; at pH4.0 to pH12 .0 are active, the optimum pH is between 7.5-8.0, and the optimum temperature is between 50-55°C. In the presence of 0.2-1% SDS or 10mM urea, proteinase K showed higher activity, but in the presence of common concentrations of EDTA, Triton X-100,...

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Problems solved by technology

[0005] The purpose of the present invention is to solve the problem of optimizing the codon and sequence of the proteinase K gene in the prior art to improve the activity of proteinase K. However, the workload is often large and the degree of activity improvement is limited, and there is often no great improvement. And proposed a kind of protease K multi-copy strain construction method

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