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Novel feruloyl esterase, mutant and application thereof

A ferulic acid esterase and mutant technology, applied in the field of bioengineering, can solve the problems of long growth cycle, few, oxygen intolerance and other problems of anaerobic fungi

Active Publication Date: 2020-10-09
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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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  • Novel feruloyl esterase, mutant and application thereof
  • Novel feruloyl esterase, mutant and application thereof
  • Novel feruloyl esterase, mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0041] Example: Research on the release of ferulic acid from the degradation of four feruloyl oligosaccharides by novel ferulic acid esterase and its mutants

[0042] Preparation of enzyme solution:

[0043] It is to insert 100mL containing 100ug / mL E. In the LB liquid medium of ampicillin sodium and 20ug / mL gentamycin, culture at 37°C and 225rpm / min for 6h. Subsequently, 15 mL of the bacterial liquid was transferred to 6 narrow-necked flasks containing 1 L of LB liquid medium containing 100 ug / mL ampicillin sodium, and cultured with shaking at 30° C. and 225 rpm / min. When the OD600 value of the liquid medium in the flask reached 0.4, 500uL of 1M IPTG (final concentration: 0.5mM) was added to each flask, and induction culture was continued at 12°C and 180rpm / min for 24h. Centrifuge at 5000rpm / min at 4°C for 10min, remove the supernatant and resuspend the bacterial cells with a specific buffer, use a homogenizer) to break the cell wall, centrifuge at 12000g at 4°C for 30min, ...

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PUM

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Abstract

The invention discloses novel feruloyl esterase, a mutant and an application thereof, and belongs to the field of bioengineering. The method comprises the following steps: firstly, successfully screening out a feruloyl esterase gene from anaerobic fungi Neocallimatix sp. by utilizing a genomics technology; according to the preference of a prokaryotic system codon, the GC content of a feruloyl esterase gene and the secondary structure of mRNA, and codon optimization is carried out on the feruloyl esterase gene to obtain a novel feruloyl esterase gene sequence. The enzyme has an esterase regionand a glucoside hydrolase 11 region, site-specific mutagenesis is carried out on the two regions; the obtained mutant gene is also expressed in escherichia coli; separation and purification results show that the novel feruloyl esterase and the mutant thereof are successfully expressed in escherichia coli, and the esterase activity of the mutant is far lower than that of a non-mutant, so that the strain source of the feruloyl esterase is enriched, and the feruloyl esterase has theoretical guiding significance in the aspects of improving the digestibility of feed fibers and improving the production performance of animals.

Description

technical field [0001] The invention belongs to the field of biological engineering, and relates to a novel ferulic acid esterase of anaerobic fungus Neocallimastix sp. and a mutant thereof, in particular to metagenomic screening of 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 h...

Claims

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Application Information

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Patent Type & Authority Applications(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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