Feruloyl esterase as well as mutant N.7-16 and application thereof

A technology of ferulic acid esterase and mutants, which is applied in the field of enzyme engineering, can solve the problems of long time-consuming enzyme production process and low ferulic acid esterase activity, and achieves expansion of preparation methods and acquisition methods, stable properties, and improved enzyme production. live effect

Active Publication Date: 2022-07-22
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 mutant N.7-16 and application thereof
  • Feruloyl esterase as well as mutant N.7-16 and application thereof
  • Feruloyl esterase as well as mutant N.7-16 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Preparation and purification of ferulic acid esterase

[0037] 1. Preparation of ferulic acid esterase

[0038] 1. PCR amplification reaction

[0039] 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 The gene sequence was converted into the gene sequence by Shanghai Sangon Biological Co., Ltd., and the gene sequence was codon-optimized according to the codon preference of the expressing strain Pichia pastoris. Finally, ferulate 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, the nucleotide sequences of which are respectively as SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 shown. Three pairs of amplification primers as shown in Table 1 were designed according to the nucleotide sequence of the ferulic acid esterase gene, and PCR amplification was car...

Embodiment 2

[0116] Example 2: Determination of the enzymatic properties of ferulic acid esterase

[0117] 1. Definition of ferulic acid esterase enzyme activity

[0118] Under certain temperature and pH conditions, the amount of enzyme required to hydrolyze methyl ferulate by ferulic acid esterase to produce 1 μmol of ferulic acid per minute was defined as 1 unit of enzyme activity, expressed as U. The specific enzyme activity of an enzyme refers to the number of units of enzyme activity per mg of ferulic acid esterase under specific conditions.

[0119] 2. Principle of enzyme activity assay

[0120] Ferulic acid esterase hydrolyzes methyl ferulate to form ferulic acid under certain temperature and pH conditions, and calculates the enzymatic hydrolysis of ferulic acid by measuring the change in the absorbance of methyl ferulate in the system before and after the reaction at OD=350 nm. The reduction of methyl ester, and then the activity of ferulic acid esterase.

[0121] 3. Plate scree...

Embodiment 3

[0149] Example 3: Acquisition of ferulic acid esterase mutants

[0150] The nucleotide sequences of the ferulic acid esterase N.1 gene, the ferulic acid esterase N.7 gene, and the ferulic acid esterase N.9 gene obtained in Example 1 were used as templates to compare and analyze the ferulic acid esters. The catalytic domain and substrate-binding domain of the enzyme were site-directed mutagenesis based on their amino acid sequences.

[0151] Ferulic acid esterase N.1-300 was obtained by increasing the number of disulfide bonds of ferulic acid esterase N.1 (nucleotide sequence SEQ ID No. 4), and ferulic acid esterase N.1-300 was composed of The mutant of G300C single point mutation (the amino acid sequence is shown in SEQ ID No. 13, the coding nucleotide sequence is shown in SEQ ID No. 14, the 300th amino acid is changed from glycine to cysteine, the corresponding DNA The sequence changed from GGT to TGT). Change the side chain group of carbohydrate binding module (CBM) of fer...

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Abstract

The invention discloses feruloyl esterase, a mutant N.7-16 of the feruloyl esterase and application of the feruloyl esterase. The feruloyl esterase has an amino acid sequence as shown in SEQ ID No.2 and 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, the feruloyl esterase mutant N.7-16 is obtained by increasing the number of disulfide bonds in the feruloyl esterase, changing side chain groups of a carbohydrate binding module and changing the charges of a catalytic structural domain, and compared with original feruloyl esterase, the feruloyl esterase mutant N.7-16 has the advantages that the feruloyl esterase mutant N.7-16 is obtained; the specific enzyme activity of the mutant is 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, in particular to a ferulic acid esterase and its mutant N.7-16 and application. Background technique [0002] Ferulic acid esterase is an enzyme that can hydrolyze ester bonds in methyl ferulate, oligosaccharide ferulate and polysaccharide ferulate, and then free ferulic acid. It is a carboxylic acid A subclass of ester hydrolases, also 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, so as to efficiently degrade polysaccharides and obtain trans-ferulic acid. Ferulic acid esterase can be used in feed industry and paper industry to improve the digestibility of fiber feed and help to remove lignin. Therefore, ferulic acid esterase has broad application prospects. [0003] Ferulic acid esterase in nature is widely found in plants and microo...

Claims

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Application Information

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