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Protease K mutant and construction and application of expression vector thereof

A protease and mutant technology, which is applied in the field of construction and application of proteinase K mutants and their expression vectors, can solve the problems of slow production of Candida albicans limberii, limitation of actual production of proteinase K, unfavorable industrial production, etc., and achieve improvement Screening for effects of weighing power, reduced handling, good enzyme activity

Active Publication Date: 2022-03-18
南京巨匠生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Due to the slow production of Candida albicans limberii, there are great restrictions on the actual production of proteinase K
Relevant researchers have tried to express proteinase K in E. coli, but most of them exist in the form of inclusion bodies, which is not conducive to industrial production

Method used

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  • Protease K mutant and construction and application of expression vector thereof
  • Protease K mutant and construction and application of expression vector thereof
  • Protease K mutant and construction and application of expression vector thereof

Examples

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preparation example Construction

[0043] The embodiment of the present invention provides the preparation method of the proteinase K mutant as described in any of the foregoing embodiments, which includes: cultivating the genetically engineered bacteria as described in the foregoing embodiments or the gene constructed by the construction method described in any of the foregoing embodiments Engineering bacteria to induce the expression of the proteinase K mutant; or, on the basis of the amino acid sequence shown in SEQ ID No.1, carry out at least one of the following mutations: F371C and T373P.

[0044] In addition, the embodiments of the present invention also provide proteinase K mutants as described in any of the foregoing embodiments or genetically engineered bacteria as described in the foregoing embodiments, or genetically engineered bacteria constructed by the construction method described in any of the foregoing embodiments can be hydrolyzed Application in protein or preparation of products for hydrolysi...

Embodiment 1

[0047] A proteinase K mutant, its amino acid sequence is shown in SEQ ID No.2, and its nucleic acid sequence is shown in SEQ ID No.3.

[0048] The preparation method of the above proteinase K mutant comprises the following steps.

[0049] 1. Construction of expression vector.

[0050] 1.1 Construction of single copy expression vector.

[0051] Using pPIC9k as the basic framework, the corresponding primers were designed according to the Golden gate assembly principle, as shown in Table 1. Six framework fragments were obtained by cloning with P202-2 high-fidelity polymerase from Nanjing Master Biotechnology Co., Ltd., which were named Frag1, Frag2, and Frag3. , Frag4, Frag5 and Frag6.

[0052] Table 1 Primer Sequence

[0053]

[0054]

[0055] Note: F is the upstream primer, R is the downstream primer.

[0056] The DNA fragments with high purity were recovered from the fragment gels obtained by the above clones, and Frag1 was fused with Frag2, Frag3 was fused with Frag...

Embodiment 2

[0076]A proteinase K mutant and its preparation method are roughly the same as in Example 1, the difference being that the strain screening steps are different. In this example, the strain screening is as follows: linearize the pATG9-3K plasmid vector, and transfer to In Pichia pastoris GS115 competent cells, high-copy high-expression strains were obtained by screening BMMY plates containing different concentrations of G418 and containing 1% casein, which specifically included the following steps:

[0077] The pATG9-3K plasmid vector was digested and linearized by SacI, and the target fragment was excised by 0.8% agarose nucleic acid electrophoresis, and the gel was recovered and purified;

[0078] The above-mentioned purified gene fragments were electrotransformed into Pichia GS115 competent cells, spread on MD plates and cultured at 28°C for 2-3 days, and the Pichia transformants on MD plates were transferred to cells containing different Concentration (50μg / ul~500μg / ul) G41...

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Abstract

The invention discloses construction and application of a protease K mutant and an expression vector thereof, and relates to the technical field of bioengineering, and provides the protease K mutant which has at least one of the following mutations on the basis of an amino acid sequence as shown in SEQ ID No.1: F371C and T373P. Compared with wild type protease K, the protease K mutant has better enzyme activity, and can be used for preparing the protease K mutant. And a way is provided for application and research of the protease K. In addition, the invention further provides construction of the protease K mutant high-copy expression vector, the screening capacity of high-expression thalli is improved, a large amount of bacterial screening operation is reduced, and a direction is pointed out for an industrial production route of protease K with application value.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to the construction and application of a proteinase K mutant and its expression vector. Background technique [0002] Proteinase K belongs to the class of serine proteases, which is a major class of proteases produced by Tritirachium album Limber. Because the microorganisms that can synthesize this kind of protease can grow in the environment with keratin (Kerantin) as the only carbon and nitrogen source, it is called proteinase K, and its three-dimensional structure is shown in figure 1 . Proteinase K has extremely high enzymatic activity and broad substrate specificity, and can preferentially decompose ester bonds and peptide bonds adjacent to the C-terminal of hydrophobic amino acids, sulfur-containing amino acids, and aromatic amino acids, and is often used to degrade proteins to produce short peptides . It has the typical catalytic triad Asp39-His69-Ser224 characteris...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/58C12N15/57C12N15/81C12N1/19C12P21/06C12R1/84
CPCC12N9/58C12N15/815C12P21/06C12N2800/102
Inventor 刁含文
Owner 南京巨匠生物科技有限公司
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