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Organic solvent-tolerant alkali protease

A technology resistant to organic solvents and proteases, applied in the field of protease genetic engineering, can solve the problems of industrial application gap, small improvement, and low organic solvent tolerance.

Inactive Publication Date: 2014-08-06
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although some progress has been made in the use of genetic modification to improve the organic solvent stability of enzymes in recent years, the organic solvent tolerance of enzymes is often relatively low, and the organic solvent tolerance, especially in hydrophilic organic solvents, is improved. The range is not large, and there is still a certain gap for industrial applications

Method used

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  • Organic solvent-tolerant alkali protease
  • Organic solvent-tolerant alkali protease
  • Organic solvent-tolerant alkali protease

Examples

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Embodiment 1

[0032]This example illustrates the directed evolution of the organic solvent-resistant alkaline protease gene of the present invention.

[0033] Select the described in the patent application publication number CN101215534A Bacillus licheniformi Cloning method of YP1A (CCTCC NO: M207021) gene encoding organic solvent-resistant alkaline protease Cloning of the gene encoding organic solvent-resistant alkaline protease, and designing primers for amplification according to the gene sequence of the p43 promoter in Bacillus subtilis 168 (GenBank number: K02714) The p43 promoter, the promoter p43 and the organic solvent-resistant alkaline protease gene were connected by overlapping PCR method, and finally connected with the expression vector pHY300 (gifted by Professor Li Shunpeng of Nanjing Agricultural University), and transformed into the Bacillus subtilis host WB800 (provided by Nanjing Agricultural University Professor Shunpeng Li), constructed a recombinant strain WB800-pHY300-...

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Abstract

The invention belongs to the technical field of protease gene engineering, and in particular relates to an organic solvent-resistant protease, in particular to its mutant gene and its encoded protein. It can be seen from SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence table that the organic solvent-resistant alkaline protease of the present invention has a site-directed mutation at the 385th or 436th position of the original nucleotide sequence, that is, the 24th amino acid mutation of its mature peptide It is glutamic acid or glutamine; the 41st amino acid is alanine or lysine. The tolerance to organic solvents, especially hydrophilic organic solvents, of the four encoded proteins after mutation was significantly enhanced.

Description

technical field [0001] The invention belongs to the technical field of protease gene engineering, and in particular relates to an organic solvent-resistant protease, in particular to its mutant gene and its coded protein. technical background [0002] Protease refers to a class of enzymes that can catalyze the hydrolysis of peptide bonds. Since it was tested as an additive ingredient in detergents in 1914, it has attracted widespread attention due to its important commercial use. Today, the production of protease accounts for more than 40% of the enzyme market, and is widely used in detergents, food, pharmaceuticals, leather, diagnostic reagents, sewage treatment and other fields. Proteases widely exist in all organisms such as animals, plants, fungi, and prokaryotes, among which proteases derived from microorganisms account for more than 2 / 3 of the total production of proteases. Proteases derived from microorganisms can be further divided into metalloproteases, aspartic pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/57C12N9/56C12R1/10
Inventor 何冰芳苏龙吴斌
Owner NANJING TECH UNIV