Protease with improved thermal stability as well as construction method and application thereof

A thermal stability, keratinase technology, applied in the field of genetic engineering, can solve the problems of polluting the environment, reducing the nutritional value of products, destroying amino acids, etc.

Active Publication Date: 2013-02-20
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 ...

Method used

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  • Protease with improved thermal stability as well as construction method and application thereof
  • Protease with improved thermal stability as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] The keratinase that embodiment 1 thermostability improves

[0018] The keratinase of the present invention is based on the gene sequence published by GenBank accessionnos.JX504681, and amino acid mutations occur at four sites in its mature region, specifically N122Y, N160C, A193P, and N217S, which can be chemically synthesized or site-directed mutation Amino acid substitutions were carried out at four sites in the mature region.

Embodiment 2

[0019] Construction and identification of embodiment 2 producing keratinase genetically engineered bacteria

[0020] The ker gene encoding keratinase in Example 1 was obtained by chemical total synthesis method, cloned into pMA0911, and the recombinant expression plasmid pMA 0911-kerTB containing the kerTB gene was obtained.

[0021] The recombinant plasmid pMA 0911-kerTB transformed Bacillus subtilis WB600 competent cells to obtain a genetically engineered bacterium that can grow normally on LB plates containing kanamycin (30 mg / mL), and was identified and named Bacillus subtilis WB600-pMA 0911- kerTB. .

Embodiment 3

[0022] Enzyme activity assay and protein electrophoresis of embodiment 3 recombinant bacteria

[0023] Keratinase enzyme activity assay method: Centrifuge the fermentation broth for 10min (10000×g, 4°C) to take the fermentation supernatant, absorb 200uL of appropriately diluted enzyme solution, add 300uL 0.05mol / L gly-NaOH buffer solution (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 10min, draw 200uL supernatant and transfer it to a new test tube, then add 1mL Folinol reagent and 200uL 0.5M Na 2 CO 3 Then develop color at 50 degrees for 15 minutes. The blank is to add 500uLTCA solution at the same time as adding the enzyme solution, and the filtered supernatant after the same process reaction is used as a blank. The absorbance was detected at 660nm, and according to the definition of the tyrosine standard curve, the release of 1ug of tyrosine in every 15min was defined as an e...

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Abstract

The invention discloses keratinase with improved thermal stability and application thereof, and belongs to the field of genetic engineering. The keratinase (ker) gene of bacillus licheniformis BBE11-1 is subjected to site-directed mutation by using a site-directed mutation technology and is cloned and linked to a bacillus subtilis expression vector pMA 0911; and bacillus subtilis WB600 is transformed, and a bacillus subtilis WB600-pMA 0911-kerTB capable of producing the keratinase with better thermal stability can be obtained through purification tests. The keratinase expressed by a strain at T1/2 at 60 DEG C lasts for 78min, which is improved nearby 9 folds compared with wild type keratinase at T1/2. The keratinase with improved thermal stability lays an excellent foundation for the application of keratinase.

Description

technical field [0001] The invention relates to a recombinant keratinase with improved thermostability, 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 energy, and can destroy some am...

Claims

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Application Information

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IPC IPC(8): C12N9/50C12N1/21C12N5/10C12N15/57C12N15/75C12R1/125
Inventor 陈坚张娟刘柏宏堵国成
Owner JIANGNAN UNIV
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