Keratinase mutant with improved thermal stability and preparation method thereof

A kind of keratinase mutation and keratinase technology, which is applied in the direction of botany equipment and methods, microorganism-based methods, biochemical equipment and methods, etc., can solve the problem of reducing the development and application of keratinase, poor thermal stability of keratinase, and substrate specificity. One problem is not high, to achieve the effect of improved thermal stability, improved enzyme activity, and good application prospects

Active Publication Date: 2014-09-03
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although keratinase has great application and research value, the keratinase screened from wild bacteria has poor thermal stability and low substrate specificity, which greatly reduces the development and application of keratinase.

Method used

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  • Keratinase mutant with improved thermal stability and preparation method thereof
  • Keratinase mutant with improved thermal stability and preparation method thereof
  • Keratinase mutant with improved thermal stability and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Construction of keratinase mutant P2C2T1 gene

[0024] (1) First use two pairs of primers F-NcoI-kersmf and R-XhoI-kersmf, F-NcoI-kersmd and R-XhoI-kersmd, to use Stenotrophomonas maltophilia BBE11-1 (in 2011 It was preserved in China Center for Type Culture Collection on April 3, 2010, and the preservation number is CCTCC No: M2011193) the genome was used as a template, and the keratinase genes kerSMF and kerSMD were amplified by PCR. The reaction conditions were as follows: 95°C pre-denaturation for 5 min, followed by a cycle: 98°C denaturation for 10 s, 58°C annealing for 15 s, 72°C extension for 1 min 50 s, 30 cycles; 72°C extension for 10 min. The DNA amplification enzyme used was Primer STAR from TaKaRa Company, and the formula was used according to the product manual.

[0025] Using the protein database of NCBI, through the amino acid sequence alignment of the Blast function, it is speculated that Keratinase KerSMD and KerSMF are divided into three dom...

Embodiment 2

[0032] Example 2 Cultivate recombinant bacteria to ferment and produce keratinase

[0033] Transform E.coli BL21 into E.coli BL21 with the recombinant expression vectors carrying the genes encoding mutants P1C2T2, P2C2T1, and P1C2T1 constructed according to the method of Example 1 to obtain genetically engineered bacteria expressing keratinase; Culture in LB medium of ampicillin overnight at 37°C, then insert into 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

[0034] Example 3 Purification and thermostability determination of keratinase mutants

[0035] (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.

[0036] (2) Using an AKTA protein purifier (GE Company of the United States) and a nickel column of HisTrap FF crude1ml, purify keratinase KerSMD with a purity of more than 90% and various mutants thereof from the crude enzyme solution. SDS-PAGE of keratinase see Figure 4 , the molecular weight of each protein is close to 46kDa.

[0037] (3) The purified mutant keratinase was added to 50 mM Gly-NaOH buffer solution (pH 9.0), incubated at 60° C. for different times, and the enzyme activity was determined. Taking the initial unheated keratinase as 100% residual enzyme activity, the percentage value of the enzymatic activity measured later compared to the initial enzymatic activity was taken as the residual enzyme activity. ...

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Abstract

The invention discloses a keratinase mutant with improved thermal stability and a preparation method thereof, and belongs to the field of enzyme engineering. Thermal stability and substrate specificity of the keratinase are improved by interchanging N ends or C ends of two different keratinases with higher homology from the same bacterial strain. The keratinase and the mutant thereof can effectively hydrolyze water-insoluble keratinase substrates such as feathers, wools, and the like, and can be used for leather spinning industry and feed industry.

Description

technical field [0001] The invention relates to a keratinase mutant with improved thermostability 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. Although keratinase has great application and research value, the keratinase screened from wild bacteria has poor thermal stability and low substrate specificity, which greatly reduces the development and application of keratinase. The keratinase gene reported so far mainly comes fro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/52C12N15/57C12N15/70C12N1/21C12R1/01
CPCC12N9/52
Inventor 陈坚张娟方真堵国成刘柏宏
Owner JIANGNAN UNIV
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