Ferulic acid esterase EpFAE1 and coding gene and application thereof

A technology of ferulic acid esterase and coding gene, applied in the field of genetic engineering, can solve problems such as deficiencies, and achieve the effects of enzymatic hydrolysis efficiency, high stability and high enzymatic hydrolysis efficiency.

Inactive Publication Date: 2019-05-31
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, many different types of ferulic acid esterases have been researched and reported successively, but the products such as ferulic acid enzyme preparations on the market are still insufficient

Method used

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  • Ferulic acid esterase EpFAE1 and coding gene and application thereof
  • Ferulic acid esterase EpFAE1 and coding gene and application thereof
  • Ferulic acid esterase EpFAE1 and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Cloning of embodiment 1 ferulic acid esterase EpFAE1 coding gene

[0038]Fungal culture and total RNA extraction: Take about 10 mg of the mycelial spore mixture of the Eupenicillium pumilus 4-14 strain into 50 mL of PDA liquid medium, and culture at 37° C. and 180 rpm for 4 days. Take 1 mL of the culture into a bottle of solid-state fermentation medium (L.Long, D.Ding, Z.Han, H.Zhao, Q.Lin, S.Ding, Thermotolerant hemicellulolytic and cellulolytic enzymes from Eupenicillium parvum 4-14 display high efficiency upon release of ferulic acid from wheat bran, J.Appl.Microbiol.121(2016) 422-434.), shake well and place it under 37°C and 70% humidity for static culture for 3 days. After the white mycelium was rinsed with sterile water, the water was blotted with filter paper, frozen in liquid nitrogen and stored at -70°C. Total bacterial RNA was extracted with TransZolTM Plant Kit (TransGen, Beijing).

[0039] Gene cloning: Take an appropriate amount of total RNA and use EasyS...

Embodiment 2

[0041] Example 2 Pichia pastoris expression and purification of ferulic acid esterase EpFAE1

[0042] Design and synthesize the specific primers fae1_f2 and fae1_r2 expressing ferulic acid esterase EpFAE1 respectively, as follows:

[0043] fae1_f2: 5'-CCGgaattcGCCGTTACGCAGGGCGTCTCTG-3';

[0044] fae1_r2:

[0045] 5'-CTAGtctagaTCAgtgatggtgatggtgatgCCAACTGCAAGCTCCGCTCG-3'.

[0046] The ferulic acid esterase EpFAE1 gene fragment containing no signal peptide was amplified from the plasmid containing the ferulic acid esterase EpFAE1 gene by using the pair of primers. The amplified target gene fragment was subjected to EcoRI-XbaI double digestion and ligated with the EcoRI-XbaI digested plasmid pPICZαA to obtain the gene expression plasmid pPIC-EpFae1. After the recombinant plasmid pPIC-EpFae1 was linearized with restriction endonuclease SacI and then electroporated into Pichia pastoris GS115, positive clones were screened using YPD plates containing 1M sorbitol and 100 μg / mL Zeo...

Embodiment 3

[0072] Example 3 Application of Ferulic Esterase EpFAE1 Synergistically Released Ferulic Acid in Cereal Bran

[0073] 1) Destarch treatment of cereal bran and preparation of enzyme

[0074] Wheat bran and corn bran purchased from the market were pulverized and sieved according to the literature method (Long et al., Thermotolerant hemicellulolytic and cellulolytic enzymes from Eupenicillium parvum 4-14 display high efficiency upon release of ferulic acid from wheatbran. Journal of Applied Microbiology , 2016, 121: 422-434) were subjected to de-starch treatment to obtain de-starch wheat bran and de-starch corn bran, which were sieved through an 80-mesh sieve for use. Ferulic acid esterase EpFAE1 was prepared according to the method in Example 2, and xylanase EpXYN1 was prepared according to the literature method (Long et al., Characterization of two new endo-β-1,4-xylanases from Eupenicillium parvum 4-14 and their applications for production of femloyl oligosaccharides. Applied...

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Abstract

The invention discloses ferulic acid esterase EpFAE1 and a coding gene and an application thereof.The amino acid sequence of the ferulic acid esterase EpFAE1 is shown as SEQ ID NO.1, and the DNA sequence of the coding gene of the ferulic acid esterase EpFAE1 is shown as SEQ ID NO.2.According to the ferulic acid esterase EpFAE1, a coding gene of the ferulic acid esterase EpFAE1 is cloned from finepenicillium 4-14, and a recombinant vector of the ferulic acid esterase EpFAE1 is expressed by Pichiapastoris to obtain pure enzyme.Experiments prove that the ferulic acid esterase EpFAE1 has the advantages of high specific activity, high enzymolysis efficiency, high stability; certain metal ion resistance, salt resistance and the like, and have an important application prospect in the fields of medicine, health care, food, feed, biological energy and the like.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a ferulic acid esterase EpFAE1, its coding gene and its application. Background technique [0002] Ferulic acid (FA), also known as 4-hydroxy-3-methoxycinnamic acid, is a phenolic acid widely present in plant cell walls. In plant cell walls, ferulic acid can be cross-linked with hemicellulose polysaccharides in the form of ester bonds, or with lignin in the form of ether bonds, forming a complex network structure. The presence of ferulic acid enhances the mechanical strength of the cell wall, which plays a very important role in preventing enzymatic degradation and protecting the integrity of the cell wall structure. Due to the specific molecular structure, ferulic acid has excellent antioxidant properties, and is used as an antioxidant in the field of medicine, and can be a precursor for the synthesis of new drugs. At the same time, ferulic acid has ant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/11C12P7/42
CPCC12N9/18C12P7/42C12Y301/01073
Inventor 龙良鲲吴莲秋丁少军
Owner NANJING FORESTRY UNIV
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