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Genetically engineered protease K and production method of protease K

A technology of genetic engineering and production method, which is applied in the field of genetic engineering proteinase K and its production, and can solve the problems of shortened production cycle and long cycle.

Inactive Publication Date: 2015-11-25
天津旭晨科技有限公司
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

Domestic proteinase K products mainly rely on imports and direct extraction from fungi. Direct extraction takes a long period, and it takes about 20 days at the current level of technology; gene recombination expression requires a lot of research and development in the early stage. If it can be expressed successfully, it can be The production cycle is shortened to 3-4 days

Method used

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  • Genetically engineered protease K and production method of protease K
  • Genetically engineered protease K and production method of protease K
  • Genetically engineered protease K and production method of protease K

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Embodiment Construction

[0014] In order to make the objects and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

[0015] 1. Obtaining the target gene

[0016] Using gene recombination technology and through gene synthesis and mutation transformation, a high-efficiency expression vector was constructed;

[0017] Amino acid mutation points include: N95C, P97S, S107D, S123A, I132V, E138A, M145F, Y151A, V167I, L180I, Y194S, A199S, K208H, A236V, R237N, P265S, V267I, S273T, G293A, L232R, S299C, I33 ;

[0018] Wherein: N in N95C is the original amino acid, 95 is the amino acid position in the protein sequence, and C is the mutated amino acid);

[0019] The proteinase K amino acid sequence, namely SEQ ID NO.1 is:

[0020] MRLSVLLSLLPLALGAPAVEQRSEAAPLIEARGEMVANK...

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Abstract

The invention discloses genetically engineered protease K. Enzymatic activity of the protease K is at least two times that of conspecific natural protease, production period can be shortened to 3-4 days, and production efficiency is greatly improved. The production method comprises the following steps of performing synthesis and amplification to obtain protease K genes; cloning and transforming to integrate the protease K genes to yeast artificial chromosome; selecting a high-quality bacterial strain capable of expressing target proteins effectively by screening yeast; performing high-density fermentation to express the protease K by using a high-density fermentation technology; and performing separation and purification on protein to obtain the high-purity and high-activity protease K.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a genetically engineered protease K and a production method thereof. Background technique [0002] At present, there are two main ways for the production of proteinase K, one is directly extracted from fungi, and the other is expressed through gene recombination. Domestic proteinase K products mainly rely on imports and direct extraction from fungi. Direct extraction takes a long period, and it takes about 20 days at the current level of technology; gene recombination expression requires a lot of research and development in the early stage. If it can be expressed successfully, it can be The production cycle is shortened to 3-4 days. From the perspective of long-term development and large-scale production, expression is preferably performed after gene recombination. Therefore, it is necessary to adopt gene recombination technology and construct a high-efficiency expression vector t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/58C12N15/81C12R1/84
Inventor 赵旭
Owner 天津旭晨科技有限公司
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