Heat-resistant cutinase-CBD (cellulose-binding domain) fusion enzyme, its mutants and application

A cutinase, mutant technology, applied in the field of genetic engineering and enzyme engineering

Active Publication Date: 2012-02-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This binding site modification strategy to improve enzyme ca

Method used

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  • Heat-resistant cutinase-CBD (cellulose-binding domain) fusion enzyme, its mutants and application
  • Heat-resistant cutinase-CBD (cellulose-binding domain) fusion enzyme, its mutants and application
  • Heat-resistant cutinase-CBD (cellulose-binding domain) fusion enzyme, its mutants and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1 The preparation method of cutinase-CBD fusion enzyme

[0017] The fusion gene encoding cutinase-CBD is obtained by the method of total gene synthesis, and the sequence is as SEQ ID NO: 1. The expression vector of cutinase-CBD gene is plasmid pET20b(+), with E. coli BL21(DE3) was used as the expression host to achieve high-efficiency expression of the thermostable cutinase-CBD gene. Its fermentation and purification methods are the same as those of the cutinase-CBD mutant.

Embodiment 2

[0018] Example 2 Effect of cutinase-CBD fusion enzyme on cotton fiber

[0019] In the 10 mL reaction system, add 1 mL cutinase-CBD (or natural cutinase) enzyme solution (enzyme activity 100 U / mL), 1 mL pectin enzyme solution (enzyme activity 100 U / mL), 8 mL 25 mM potassium phosphate (pH 8.0), 0.5g raw cotton fiber and 50μL penetrant TX-10. The control was the same as the above conditions but no cotton fiber was added to the treatment solution. React at room temperature and take samples regularly. After the sample was centrifuged for 3 min, the enzyme activity in the supernatant was measured according to the method for measuring enzyme activity. The amount of enzyme adsorbed is the enzyme activity in the control solution minus the enzyme activity in the supernatant. The results showed that under the interaction with pectinase, the adsorption rate of natural cutinase to cotton fiber was 10%, while the adsorption rate of cutinase-CBD to cotton fiber was 55%.

[0020] The 10...

Embodiment 3

[0021] Example 3 Determination of cutinase-CBD mutant mutation site

[0022] The key amino acid for the cellulose binding domain to bind to cellulose is the highly conserved tryptophan residue. Through the multiple sequence alignment of the second family CBD, combined with the structural characteristics of polyester fibers, two key points were mutated into amino acids similar to polyester polyester structures from the above two aspects to enhance the hydrophobic interaction between the enzyme and the substrate, thereby Improve catalytic efficiency. The specific mutations are as follows: (1) mutate tryptophan to leucine to enhance the hydrophobic interaction with the alkyl chain in the polyester structure to improve the affinity with the substrate; (2) mutate tryptophan to tyrosine Amino acid improves the hydrophobic interaction with the benzene ring in the polyester structure, and the hydroxyl group generates hydrogen bonds to enhance the binding force with the substrate. ...

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Abstract

Belonging to the genetic engineering and enzyme engineering fields, the invention discloses a heat-resistant cutinase-CBD fusion enzyme, its mutants and application in polyester fiber modification. The invention provides a preparation method of a Thermobifidafusca cutinase-CBD fusion enzyme and its mutants. The mutants comprise a substitute at a binding site on the CBD of the Thermobifidafusca cutinase-CBD fusion enzyme. Adsorption efficiency of mutants W68L and W68Y for polyester fiber are enhanced by 14% and 17% respectively compared with the cutinase-CBD fusion enzyme, while the hydrolysisabilities of the mutants for polyester fiber is 1.4 and 1.5 times of the hydrolysis ability of the cutinase-CBD fusion enzyme. And the two mutants of the cutinase-CBD fusion enzyme are more suitable for application in polyester fiber modification.

Description

technical field [0001] The invention relates to a heat-resistant cutinase-CBD fusion enzyme and its mutant and its application in polyester fiber modification, belonging to the fields of genetic engineering and enzyme engineering. Background technique [0002] As one of the three major synthetic fibers in the world today, polyester exhibits good physical properties (such as strength, elasticity, toughness, hardness and abrasion resistance, etc.), dyeing performance, high temperature resistance, and chemical resistance compared with natural fibers. characteristic. However, since polyester fiber is a hydrophobic fiber, it has high crystallinity, poor hygroscopicity, and is prone to static electricity, which not only affects wearing comfort, but also limits the processing of compound modifiers, colorants, flame retardants, etc. in the application. Therefore, in order to achieve excellent wettability and dyeing effect, polyester fiber must be modified. At present, the tradit...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/62D06M16/00D06M101/32
Inventor 吴敬陈晟张瑶吴丹陈坚夏泽华
Owner JIANGNAN UNIV
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