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Feruloyl esterase as well as mutants and application thereof

A technology of ferulic acid esterase and mutant, which is applied in the field of enzyme engineering, can solve the problems of low ferulic acid esterase activity and long time-consuming process of producing enzyme, achieve stable properties, expand preparation methods and acquisition methods, and improve enzyme production. live effect

Active Publication Date: 2021-06-18
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the activity of ferulic acid esterase secreted by the wild-type strain is relatively low and the enzyme production process takes a long time, so it is necessary to obtain a new type of ferulic acid esterase with higher enzyme activity through abundant genetic resources

Method used

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  • Feruloyl esterase as well as mutants and application thereof
  • Feruloyl esterase as well as mutants and application thereof
  • Feruloyl esterase as well as mutants and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: Preparation and purification of ferulic acid esterase

[0039] One, the preparation of ferulic acid esterase

[0040] 1. PCR amplification reaction

[0041] The amino acid sequences of ferulic acid esterase N.1, ferulic acid esterase N.7 and ferulic acid esterase N.9 (respectively shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 shown) was transferred to the gene sequence by Shanghai Sangon Biotechnology Co., Ltd., and the gene sequence was codon-optimized according to the codon preference of the expression strain Pichia pastoris, and finally ferulic acid esters with lengths of 1065bp, 1203bp and 864bp were obtained Enzyme N.1 gene, ferulic acid esterase N.7 gene, ferulic acid esterase N.9 gene, their nucleotide sequences are respectively as SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 shown. According to the nucleotide sequence of the ferulic acid esterase gene, 3 pairs of amplification primers as shown in Table 1 were designed, and PCR amplification was carrie...

Embodiment 2

[0119] Embodiment 2: Determination of ferulic acid esterase enzymatic properties

[0120] 1. Definition of ferulic acid esterase enzyme activity

[0121] Under certain temperature and pH conditions, the amount of enzyme needed to hydrolyze methyl ferulate to produce 1 μmol of ferulic acid per minute by ferulic acid esterase is defined as 1 enzyme activity unit, expressed in U. The specific enzyme activity of an enzyme refers to the number of enzyme activity units per mg of ferulic acid esterase under specific conditions.

[0122] 2. Principle of Enzyme Activity Determination

[0123] Ferulic acid esterase hydrolyzes ferulic acid methyl ester to generate ferulic acid under certain temperature and pH conditions, and calculates the enzymatic hydrolysis of ferulic acid by measuring the change of absorbance of ferulic acid methyl ester in the system before and after the reaction at 0D=350nm The reduction of methyl ester, and then the activity of ferulic acid esterase.

[0124] 3...

Embodiment 3

[0152] Embodiment 3: the acquisition of ferulic acid esterase mutant

[0153] With the nucleotide sequence of ferulic acid esterase N.1 gene, ferulic acid esterase N.7 gene, ferulic acid esterase N.9 gene that embodiment 1 obtains respectively as template, comparative analysis of ferulic acid ester The catalytic domain and substrate binding domain of the enzyme were subjected to site-directed mutagenesis according to its amino acid sequence.

[0154]Increase the number of disulfide bonds of ferulic acid esterase N.1 (nucleotide sequence SEQ ID No.4) to obtain ferulic acid esterase N.1-300, ferulic acid esterase N.1-300 is containing The mutant of G300C single point mutation (amino acid sequence is shown in SEQ ID No.13, and the coding nucleotide sequence is shown in SEQ ID No.14, the 300th amino acid is changed from glycine to cysteine, and its corresponding DNA sequence changed from GGT to TGT). Change the side chain group of the carbohydrate binding module (CBM) of ferulic...

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Abstract

The invention discloses feruloyl esterase as well as mutants and application thereof. The feruloyl esterase has an amino acid sequence as shown in SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3, the feruloyl esterase is stable in property, the reaction pH is 6-8, and the reaction temperature is 37-54 DEG C. On the basis of the amino acid sequence of the feruloyl esterase, three feruloyl esterase mutants N.1-300, N.7-16 and N.9-98 are obtained by increasing the number of disulfide bonds in the feruloyl esterase, changing side chain groups of a carbohydrate binding module of the feruloyl esterase and changing the charges of a catalytic structural domain, and compared with original feruloyl esterase, the specific enzyme activities of the three mutants are obviously improved. The feruloyl esterase and the mutant obtained by the invention have stable properties, and are beneficial to industrial application of the feruloyl esterase.

Description

technical field [0001] The invention belongs to the field of enzyme engineering, and in particular relates to a ferulic acid esterase and its mutant and application. Background technique [0002] Ferulic acid esterase is an enzyme that can hydrolyze the ester bonds in ferulic acid methyl ester, oligosaccharide ferulic acid ester and polysaccharide ferulic acid ester, and then release ferulic acid. It is a carboxylic acid A subclass of esterhydrolases and an extracellular enzyme. At present, in the food industry, ferulic acid esterase is usually used to break the cross-linking of ferulic acid and polysaccharides in cell wall materials such as bran and straw, efficiently degrade polysaccharides and obtain trans-ferulic acid. In the feed industry and paper industry, ferulic acid esterase can be used to improve the digestibility of fiber feed and help remove lignin. Therefore, ferulic esterase has broad application prospects. [0003] Ferulic acid esterase in nature widely ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/55
CPCC12N9/18C12Y301/01073C12P7/42Y02P60/87
Inventor 成艳芬马玉萍朱伟云
Owner NANJING AGRICULTURAL UNIVERSITY
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