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A kind of low temperature alkaline protease and preparation method thereof

A kind of protease and alkaline technology, applied in the field of bioengineering, can solve the problems that are not suitable for the development of enzyme-added detergents

Active Publication Date: 2011-12-14
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the enzyme activity of traditional detergent enzymes under low temperature conditions is no longer suitable for the development of a new generation of enzyme-added detergents.

Method used

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  • A kind of low temperature alkaline protease and preparation method thereof
  • A kind of low temperature alkaline protease and preparation method thereof
  • A kind of low temperature alkaline protease and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0029] 1. Obtaining the wild-type alkaline protease mature peptide gene Mapr

[0030] (1) Extraction of chromosomal DNA from Bacillus alcalophilius ATCC 31408:

[0031] ①Put 1mL of the bacterial solution in a 1.5mL Ep tube, centrifuge at 12000r / min for 1min, discard the supernatant, and collect the bacterial cells.

[0032] ②Suspend in 100 μL of pH 8.0 TE buffer solution containing lysozyme (2 mg / mL), pipette repeatedly with a pipette tip evenly, and place in a 37°C incubator for 30-60 minutes.

[0033] ③ Add 400 μL of lysate, quickly pipette with the pipette tip, and mix well.

[0034] ④Add 200μL 5mol / L NaCl, mix lightly, and centrifuge at 12000r / min for 10min.

[0035] ⑤ Transfer the supernatant to another sterilized Ep tube, add an equal volume of phenol: chloroform = 1: 1, invert gently several times, and mix well.

[0036] ⑥Centrifuge at 12000r / min for 10min, then take the supernatant, extract twice with phenolform, and finally extract once with an equal volume of chlo...

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Abstract

The invention relates to a low-temperature alkaline protease and a preparation method thereof, belongs to a method of performing site-directed mutagenesis to a wild type alkaline protease gene by utilizing a recombinant DNA (deoxyribonucleic acid) technology to improve the characteristics of the gene, connecting the mutated gene with escherichia coli-bacillus subtilis shuttle plasmid pBE2S and expressing the mutated gene in the bacillus subtilis at high efficiency, and relates to a low-temperature alkaline protease with cold adaptability and alkali stability and a preparation method thereof. The invention solves the problem that the alkaline protease has so low activity in a low-temperature environment that the application is limited. The invention adopts a technical scheme that: the wildtype alkaline protease gene is separated from a microbe, in particular bacillus alcalophilus; amino acid residues of Glu110 and Glu134 of the gene are mutated; the enzyme activity of fermentation fluid of the gene is 1985 U / mL after the gene is expressed in the bacillus subtilis at high efficiency; at 40 DEG C, the activity of the low-temperature alkaline protease (Glu110 and 134 Ala) is improvedby 28 percent, compared with that of the wild type alkaline protease; and at 10 DEG C, the activity is improved by 62 percent, compared with that of the wild type alkaline protease.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to site-directed mutation and recombination technology of genes, in particular to a low-temperature alkaline protease and a preparation method thereof. Background technique [0002] In the early 1970s, my country successfully developed alkaline protease and realized industrial production. The industrial production strain is a mutant strain of Bacillus licheniformis 2709, but the enzymatic properties of alkaline protease 2709 are poor, such as: heat resistance, reaction pH range, substrate specificity, safety issues, etc. The annual output of alkaline protease is very low, and the vast majority of alkaline protease manufacturers have stopped production. [0003] At present, domestic detergent manufacturers mainly use enzymes from Danish Novoenzyme Company and American Genencor Company. At present, the total output of detergents in my country is 2.7 million tons, while the production of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/54C12N15/57C12N15/75C12R1/07C12R1/125
Inventor 路福平刘逸寒刘敏尧刘靓薄嘉鑫王春霞王建玲
Owner TIANJIN UNIV OF SCI & TECH
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