Keratinase with improved thermal stability and specific activity as well as preparation method and application thereof

A technology of keratinase and heat stability, applied in the field of genetic engineering, can solve problems such as polluting the environment, reducing the nutritional value of products, and large energy consumption

Active Publication Date: 2014-02-26
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thermal degradation and other methods, the former has the problem of polluting the environment, the latter consumes a lot of energy, and can destroy some amino acids, reducing the nutritional value of the product
Other common keratin is animal hair, such as cow hair, wool, and human hair. Some of these keratin proteins are processed as commercial raw materials, and the rest are mostly treated as waste, which also causes environmental pollution and waste of protein resources.

Method used

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  • Keratinase with improved thermal stability and specific activity as well as preparation method and application thereof
  • Keratinase with improved thermal stability and specific activity as well as preparation method and application thereof
  • Keratinase with improved thermal stability and specific activity as well as preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The keratinase that embodiment 1 thermostability improves

[0024] The keratinase of the present invention is based on the gene sequence published by GenBank accession nos. JX504681, and carries out molecular transformation in the cleavage site region of its leader peptide, specifically the C-terminal (-1) site Leu of the leader peptide is replaced with Ala, and the mature The N-terminal AQTVP of the enzyme was replaced by the N-terminal YTPNDPYFSSRQ of the thermophilic protease derived from Thermoactinomyces vulgaris. The method of obtaining it is to use the gene published by GenBank accession nos. JX504681 as the starting gene, and carry out amino acid substitution in the cleavage site region of the leading peptide by chemical total synthesis or site-directed mutagenesis; the amino acid sequence is shown in SEQ ID NO.1.

Embodiment 2

[0025] Construction and identification of embodiment 2 producing keratinase genetically engineered bacteria

[0026] The steps of constructing keratinase genetically engineered bacteria are as follows:

[0027] 1) The ker gene is obtained by PCR method or chemical total synthesis method;

[0028] 2) Link the ker gene to the cloning vector pMD19-T to obtain the recombinant vector pMD19-T-ker;

[0029] 3) Leu at the C-terminal (-1) site of the leader peptide was replaced with different types of amino acids. The N-terminal AQTVP of the mature enzyme was replaced by the N-terminal YTPNDPYFSSRQ (N-YPTND) of a thermophilic protease derived from Thermoactinomyces vulgaris.

[0030] The mutation can adopt site-directed mutagenesis technique or other techniques, as long as the mutation can be realized.

[0031] This patent lists the primers of Leu(-1)Ala as an example:

[0032] Leu(-1)Ala: GATCATGTGGCCCATGCC TTG (GCA)GCGCAAACCGTTCCTTACN-YPTND:

[0033] TATACGCCTAATGACCCTTATTTC...

Embodiment 3

[0047] Enzyme activity assay and protein electrophoresis of embodiment 3 recombinant bacteria

[0048] Keratinase activity assay method: Centrifuge the fermentation broth for 10min (10000×g, 4°C) to take the fermentation supernatant, absorb 200μL of appropriately diluted enzyme solution, add 300μL of 0.05mol / L gly-NaOH buffer (pH9.0) Dissolve 1% of the substrate (keratin), incubate at 50°C for 20min, add 500uL 4M TCA solution to terminate the reaction. Centrifuge for 10 minutes, pipette 200 μL of supernatant into a new test tube, and then add 1 mL of folinol reagent and 200 μL of 0.5M Na 2 CO 3 Then develop color at 50 degrees for 15 minutes. For the blank, 500 μL of TCA solution was added at the same time as the enzyme solution was added, and the filtered clear liquid reacted through the same process was used as a blank. The absorbance was detected at 660nm, and according to the definition of the tyrosine standard curve, 1 μg of tyrosine was released every 15 minutes, whic...

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Abstract

The invention discloses keratinase with improved thermal stability and specific activity as well as a preparation method and application thereof, and belongs to the field of genetic engineering. The keratinase having wide industrial application prospect is obtained by mutating a leading peptide cleavage site of keratin and transforming the N-terminal of the keratin. The t1 / 2 of the keratinase at the temperature of 60 DEG C is 20 minutes and is increased by nearly two times in comparison with that of wild type keratinase. Meanwhile, the specific activity of a keratinase mutant is increased by nearly 50% in comparison with that of the wild type keratinase, so that a good foundation is laid for the application of the keratinase.

Description

technical field [0001] The invention relates to a recombinant keratinase with improved specific enzyme activity and thermal stability, its construction method and application, and belongs to the technical field of genetic engineering. Background technique [0002] Keratinase is an enzyme that can specifically degrade keratin, which is produced by various microorganisms such as bacteria, actinomycetes and fungi. As a by-product of poultry breeding and slaughtering industry, feathers have a huge annual output and are rich in protein and amino acids, so they are potential excellent protein resources. Reasonable utilization of feathers can reduce the pollution of waste to the environment on the one hand, and can be used as a feed protein for livestock and poultry feeding. In the traditional process of using feathers, acid-base hydrolysis is mainly used. Thermal degradation and other methods, the former has the problem of polluting the environment, the latter consumes a lot of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/56C12N15/57C12N15/75C12N1/21C12R1/125
CPCC12N9/54
Inventor 陈坚刘柏宏张娟堵国成
Owner JIANGNAN UNIV
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