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Keratinase mutant with improved catalytic performance and application

A kind of keratinase mutation, keratinase technology, applied in the directions of application, hydrolase, biochemical equipment and method, can solve the problems of high production cost, low enzyme production level and the like, and achieve the effect of good thermal stability

Inactive Publication Date: 2019-04-09
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still few potential strains capable of effectively expressing highly active keratinase, and keratinase-producing bacteria generally have the problems of low enzyme production levels and high production costs, which will become the main obstacle to the industrial production and application of keratinase

Method used

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  • Keratinase mutant with improved catalytic performance and application
  • Keratinase mutant with improved catalytic performance and application
  • Keratinase mutant with improved catalytic performance and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: the method for expressing KerBp in Bacillus subtilis system

[0027] Use gene-specific primers designed according to the coding sequence (SEQ ID NO.1) to amplify KerBp from the genome of Bacillus pumilus, connect the gel-purified PCR product to the pMD19-T simple vector, and then transform the ligated product into The clone host was Escherichia coli JM109, and cultured on LB agar plates containing ampicillin resistance (100 μg / mL) for 12 hours at 37°C. Positive colonies were inoculated into 10 mL of liquid LB containing ampicillin resistance (100 μg / mL) and cultured for 10-12 hours, plasmids were extracted and verified by DNA sequencing (Sangon Biotech). The cloning plasmid and expression vector pMA5 were digested with restriction endonucleases BamHI and Mlu I, then ligated with T4 DNA ligase at 16°C for 8 hours to construct the expression plasmid kerBp-pMA5, and transformed into the cloning host Escherichia coli JM109 by heat shock method In, the express...

Embodiment 2

[0028] Embodiment 2: fermentation culture condition and keratinase assay method

[0029] The recombinant strain was cultured at 37°C for 12h in 10 mL of LB medium containing kana resistance (100 μg / mL). Subsequently, the seed solution was inoculated into 50 mL of TB medium containing kana resistance (100 μg / mL), and further cultured at 37° C. for 48 h. Bacillus subtilis WB600 carrying pMA5 (used as a control group) was also cultured under the same conditions. The fermentation broth was centrifuged at 12,000 rpm for 5 min, and the supernatant was collected for activity determination and SDS-PAGE analysis.

[0030] Enzyme activity was determined at pH 9.0 and 40°C using 1% soluble keratin as substrate. The reaction mixture contained 1 mL of enzyme and 1 mL of 1% soluble keratin diluted in Tris-HCl buffer (0.05M, pH 9.0). The reaction was carried out in a water bath at 40° C. for 15 min, and the reaction was terminated with 2 mL of 5% TCA. The mixture was centrifuged at 12000r...

Embodiment 3

[0031] Example 3: Verification of the role of the leader peptide

[0032] Using the plasmid kerBp-pMA5 as a template, the signal and mature keratinase were amplified by primers Signal(F) / Signal(D) and ker(F) / ker(D), respectively, to construct the leader peptide-deficient expression plasmid SMF-pMA5. Carry out cloning in Escherichia coli JM109, then extract plasmid, express in Bacillus subtilis WB600, through SDS-PAGE analysis, observe that molecular weight is the protein band of 30kDa, but do not detect keratinase activity ( figure 2 a).

[0033] Using the kerBp-pMA5 plasmid as a template, the signal peptide sequence and the truncated mutant sequence were respectively amplified by designing primers with different homology arms, and then the signal peptide was added to the N-terminus of the mature enzyme to obtain recombination with different leader peptide lengths Plasmids, expressed in Bacillus subtilis WB600, and assayed for activity. The results of the activity assay sho...

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Abstract

The invention discloses a keratinase mutant with improved catalytic performance and an application, belongs to the technical field of genetic engineering and provides recombinant keratinase constructed on the basis of leading peptide engineering and multi-site saturated mutation and a leading peptide engineering method. A recombination strain M7 shows high keratinase catalytic potential, enzyme activity is remarkably improved from 179 U / mL to 1114 U / mL, the optimal reaction temperature is 40 DEG C, the optimal reaction pH is 10.0, over 80% enzyme activity is retained after water bath processing is performed for 90 min at 40 DEG C, heat stability is better, and the keratinase mutant is suitable for industrial large-scale production. Besides, the method for modifying protease with the leading peptide engineering can be a universal way for modifying industrial enzyme with the self-shearing characteristic.

Description

technical field [0001] The invention relates to a keratinase mutant with improved catalytic performance and its application, belonging to the technical field of genetic engineering. Background technique [0002] Keratin is the structural protein of ectoderm cells, a kind of rigid fibrous protein, containing more hydrogen bonds and disulfide bonds, and its three-dimensional structure has high stability, coupled with hydrophobic effect, it is difficult to be digested by trypsin, pepsin, papaya Efficient degradation by protease etc. The protein in keratin is rich in nutrients, the protein in feather accounts for 80%, and the wool can account for 99%, and it contains the essential amino acids needed by various animals. These have high application value, but these protein resources are widely used. Waste, but also caused pollution to the environment, so the recycling and utilization of keratin has been paid more and more attention by people. [0003] Keratinase has a specific f...

Claims

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Application Information

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IPC IPC(8): C12N9/52C12N15/55C12N15/75C12N15/70C12N1/21
CPCC12N9/52C12N15/70C12N15/75
Inventor 史劲松龚劲松苏畅许正宏李恒孙雨欣
Owner JIANGNAN UNIV
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