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A kind of ferulic acid esterase, mutant and application

A technology of ferulic acid esterase and esterase, which is applied in the field of bioengineering, can solve the problems of oxygen intolerance, long and few anaerobic fungal growth cycles, etc., so as to improve digestibility, enrich the source of bacteria, and improve animal health. The effect of production performance

Active Publication Date: 2022-05-20
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, due to the long growth cycle of anaerobic fungi and extreme intolerance to oxygen, the expression and enzymatic characterization of anaerobic fungal ferulic acid esterase are still rare.

Method used

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  • A kind of ferulic acid esterase, mutant and application
  • A kind of ferulic acid esterase, mutant and application
  • A kind of ferulic acid esterase, mutant and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0041] Example: Ferulic acid esterase and its mutants degrade four feruloyl oligosaccharides to release ferulic acid

[0042] Preparation of enzyme solution:

[0043] The recombinant bacteria containing the gene encoding ferulic acid esterase or its mutant (Escherichia coli (DE3) containing the gene of ferulic esterase or its mutant) were activated and cultured, and then inserted into 100mL containing 100ug / mL ampicillin sodium and 20ug / mL gentamycin in LB liquid medium, cultured at 37°C and 225rpm / min for 6h. Then 15mL of bacterial liquid was transferred to 6 LB containing 1L containing 100ug / mL ampicillin sodium In the narrow-necked bottle of liquid medium, shake culture under the condition of 30 ℃, 225 rpm / min. When the OD600 value of the liquid medium in the narrow-necked bottle reaches 0.4, add 500uL 1 M IPTG in each bottle ( The final concentration is 0.5 mM), 12°C, 180 rpm / min to continue the induction culture for 24h. 5000rpm / min, 4°C centrifuge for 10min, remove the ...

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Abstract

The application discloses a ferulic acid esterase, a mutant and its application, belonging to the field of bioengineering. The present invention first utilizes genomics technology to successfully screen out a ferulic acid esterase gene from the anaerobic fungus Neocallimastix sp., according to the preference of prokaryotic system codon usage and the GC content of the ferulic esterase gene and the binary content of the mRNA. The codon optimization of the ferulic acid esterase gene was carried out to obtain the gene sequence of the ferulic acid esterase. The enzyme has an esterase region and a glycoside hydrolase region 11, and these two regions are subjected to site-directed mutation, and the mutant gene obtained is also expressed in Escherichia coli, and the results of separation and purification show that the ferulic acid esterase and its mutants are successfully Expressed in Escherichia coli, and the esterase activity of the mutant is much lower than that of the non-mutant, which enriches the strain source of ferulic acid esterase, and has theoretical guidance in improving the digestibility of feed fiber and improving animal production performance significance.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to a ferulic acid esterase of the anaerobic fungus Neocallimastix sp. and a mutant thereof, in particular to the metagenomic screening of the anaerobic fungal Neocallimastix sp., ferulic acid esterase gene codon optimization, Expression and application of ferulic esterase and its mutants. Background technique [0002] The composition of straw-based roughage is generally based on cellulose, hemicellulose and lignin, and also includes some phenolic acids such as ferulic acid, coumaric acid and sinapinic acid linked by ester bonds or ether bonds. There will be some ferulic acid on the side chain of hemicellulose (arabinoxylan), which will affect the effective degradation of hemicellulose by hemicellulase. Let the hemicellulase fully exert its degrading effect. Ferulic esterase, mainly derived from bacteria and some aerobic fungi such as Aspergillus niger, is a subclass of carboxylate hydr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/70C12P19/00
CPCC12N9/18C12N15/70C12P19/00C12Y301/01073C12N2800/22Y02P60/87
Inventor 成艳芬施其成李袁飞朱伟云
Owner NANJING AGRICULTURAL UNIVERSITY
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