Catalytic rate improving keratinase mutants and preparation method thereof

A technology of keratinase mutation and keratinase, applied in biochemical equipment and methods, botanical equipment and methods, hydrolytic enzymes, etc. Detergent and other problems, to achieve the effect of enhancing the anti-detergent SDS ability and good application prospects

Active Publication Date: 2015-10-28
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although keratinase has great application and research value, the keratinase screened from wild bacteria has low catalytic efficiency,

Method used

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  • Catalytic rate improving keratinase mutants and preparation method thereof
  • Catalytic rate improving keratinase mutants and preparation method thereof
  • Catalytic rate improving keratinase mutants and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Preparation of recombinant bacteria expressing keratinase C-terminal shear mutants

[0028] (1) According to the special β-sheet structure at the C-terminus, determine the cut site such as figure 1 . Then use a pair of primers (Table 1) carrying double restriction sites NcoI and XhoI, to contain Stenotrophomonas maltophilia (Stenotrophomonas maltophilia) BBE11-1 (on April 3, 2011 preserved in Chinese Type Culture The depository center, the deposit number is CCTCC No: M 2011193) The plasmid pET22b+kerSMD of the keratinase gene kerSMD was used as a template for PCR amplification. The reaction conditions are as follows: 95°C pre-denaturation for 5 minutes, followed by a cycle: 98°C denaturation for 10s, 55°C annealing for 10s, 72°C extension for 7min 50s, 30 cycles; 72°C extension for 1min 50s, and then cooling down to 12°C to obtain the final reaction solution . The DNA amplification enzyme used was Primer STAR from TaKaRa Company, and the formula was used ac...

Embodiment 2

[0034] Embodiment 2 recombinant bacterium fermentation produces keratinase mutant

[0035] Transform E.coli BL21 into E.coli BL21 with the recombinant expression vectors constructed according to the method in Example 1 to obtain the genetically engineered bacterium expressing keratinase; Medium 37°C liquid culture overnight, then insert LB fermentation liquid medium containing 100 μg / l ampicillin and culture at 37°C until OD 600 = 0.6, lower the temperature to 20°C for culture, add the inducer IPTG with a final concentration of 0.1 mM to induce culture, and centrifuge at 72 hours to obtain the supernatant enzyme solution, which is the crude enzyme solution.

Embodiment 3

[0036] Purification of embodiment 3 keratinase mutants and specific enzyme activity and catalytic rate determination

[0037](1) The recombinant Escherichia coli containing the plasmid of the mutant gene was induced and cultured at 20° C. for 3 days to obtain a crude enzyme solution.

[0038] (2) Purify keratinase KerSMD with a purity of more than 90% and various mutants thereof from the crude enzyme solution by using an AKTA protein purifier (GE Company of the United States) and a nickel column of HisTrap FF crude 1 ml. SDS-PAGE of keratinase see figure 2 , the molecular weight of each protein is close to 43kDa.

[0039] (3) The purified mutant keratinase was added to 50 mM Gly-NaOH buffer (pH 9.0) containing 1% (w / v) casein, incubated at 50° C. for different times, and the enzyme activity was determined. And the catalytic rate and other reaction kinetic parameters of the keratinase mutants to the synthetic substrate AAPF were determined.

[0040] (4) It can be seen from ...

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Abstract

The present invention discloses catalytic rate improving keratinase mutants and a preparation method thereof, and belongs to the field of enzyme engineering. According to the present invention, the C terminal structure of keratinase KerSMD is sheared to obtain a series of keratinase mutants, wherein the catalytic rate of the keratinase mutants on the casein substrate is significantly improved and is increased by 33-103%, particularly the mutant V355 has effects of significantly improved catalytic rate, significantly improved enzyme activity, enhanced salt tolerance and enhanced detergent resistance, the characteristics of no collagenase activity and salt tolerance of the V355 can be used in the leather processing industry, the catalytic rate on the substrate is rapid, and the V355 has the characteristic of detergent resistance so as to be subjected to laundry additive production application.

Description

technical field [0001] The invention relates to a keratinase mutant with improved catalytic rate and a preparation method thereof, belonging to the field of enzyme engineering. Background technique [0002] Keratinase is an enzyme that can specifically degrade keratin, which is produced by various microorganisms such as fungi, actinomycetes and bacteria. Keratinase is widely used in industries such as food, medicine, feed, refining and tanning. It has the functions of tenderizing meat, producing advanced nutrition, immune preparations and feed additives, as well as beautifying and softening leather. It can also cause mad cow disease and human Degradation of Prion in Creutzfeldt-Jakob disease. The keratinase gene reported so far mainly comes from the kerA gene of a foreign strain of Bacillus licheniformis. Although keratinase has great application and research value, the keratinase screened from wild bacteria has low catalytic efficiency, low substrate specificity, and cann...

Claims

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Application Information

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IPC IPC(8): C12N9/52C12N15/57C12N15/70C12N1/21
CPCC12N9/52
Inventor 张娟方真堵国成陈坚
Owner JIANGNAN UNIV
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