Protease K multi-copy strain construction method

A construction method and multi-copy technology, applied in the biological field, can solve the problems of large workload, limited activity, and no great improvement, and achieve the effect of increasing yield and low price.

Pending Publication Date: 2021-02-12
CUSABIO TECH LLC
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  • Abstract
  • Description
  • Claims
  • Application Information

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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. Howeve

Method used

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  • Protease K multi-copy strain construction method

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Embodiment Construction

[0039] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention.

[0040] The present invention constructs protease K multi-copy strains in vitro by gene editing method, and after large-scale fermentation production, the final output can reach 8g / L, which can reach the highest level of current domestic output;

[0041] The following primer sequences, target gene sequences, and carrier structure biological material information will be used as specific methods for implementing the present invention.

[0042]

[0043]

[0044] A method for constructing protease K multi-copy strains, using the Biobrick method to artificially construct protease K multi-copy strains in vitro.

[0045] The specific steps are mainly as follows:

[00...

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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,...

Claims

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Application Information

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IPC IPC(8): C12N9/60C12N15/81C12N15/66C12N15/64C12N1/19C12R1/84
CPCC12N9/60C12N15/815C12N15/66C12N15/64
Inventor 杨琥陈莹谭华菊
Owner CUSABIO TECH LLC
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