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Feruloyl esterase A mutant and purpose thereof

A technology of ferulic acid esterase and mutants, which can be used in the fields of genetic engineering and enzyme engineering, and can solve problems such as unsatisfactory thermal stability

Inactive Publication Date: 2011-10-19
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Ferulic acid esterase currently used in production is mainly derived from ferulic acid esterase A of Aspergillus niger, but its thermal stability is still not ideal

Method used

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  • Feruloyl esterase A mutant and purpose thereof
  • Feruloyl esterase A mutant and purpose thereof
  • Feruloyl esterase A mutant and purpose thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Embodiment 1 Error-prone PCR (error-prone PCR) method constructs ferulic acid esterase library

[0025] use II Random Mutagenesis kit randomly mutates the ferulic acid esterase gene (see SEQ ID NO.1).

[0026] The primers used were: faeA-F: 5'-taggaggt gaattc gcctccacgcaaggcatctc-3'

[0027] faeA-R: 5'-taggaggt gcggccgc ttaccaagtacaagctccgctcg-3'

[0028] The reaction conditions were: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 61°C for 1 min and extension at 72°C for 1 min, a total of 25 cycles, and the gene fragments were recovered after electrophoresis.

[0029] The recovered fragment was double digested with NotI and EcoRI, and ligated with the pGAPZαA vector (containing the bleomycin resistance gene) that had undergone the same digestion. The molar ratio of pGAPZαA carrier digested by double enzyme digestion and the target gene fragment was mixed at a ratio of 1:3, and 600 units of T 4 Ligase, ligated overnight at 16°C. The...

Embodiment 2

[0030] The screening of embodiment 2 ferulic acid esterase mutant libraries

[0031] After collecting the secondary mutant library clones in Example 1, the plasmids were extracted and linearized with PmeI endonuclease, then electroporated to transform Pichia pastoris (Pichia pastoris KM71) competent cells and spread on MD plates (1.34% YNB, 4×10 -5% biotin, 2% glucose). After culturing for 2 days, single clones were picked and placed in a 96-well plate, each well containing 150 μl of BMGY medium (containing 100 mM, potassium phosphate buffer with a pH value of 6.0, 2% tryptone, 1% yeast extract, 1% glycerol, 4×10 -5 % biotin), 30°C, 280rpm, shake culture for 2 days. Use a 96-well plate replicator to replicate each single clone on an MD solid medium plate, culture it at 30°C for 2 days, and store it in a refrigerator at 4°C. After centrifuging the 96-well plate culture, remove the supernatant, and add 150 μl of BMMY (in addition to replacing 1% glycerol with 0.5% methanol in ...

Embodiment 3

[0032] Embodiment 3 Integration of ferulic acid esterase mutation site

[0033] The 12 mutation sites obtained above were integrated into a nucleotide coding sequence and the complete sequence was synthesized by Shanghai Sangon Biotechnology Service Co., Ltd. In order to facilitate subsequent protein purification, the synthesized full sequence was double digested with NotI and EcoRI to recover the target fragment, and ligated into the pGAPZαA vector (containing the bleomycin resistance gene) after the same digestion (the ligation method is as in Example 1) . The ligated product was transformed into Escherichia coli DH5α, and the transformant was picked and cultured to extract the recombinant plasmid. After the plasmid was linearized with BspHI endonuclease, it was transformed into Pichia pastoris (Pichia pastoris KM71) competent cells, and the YPDS medium containing 100 μg / ml bleomycin (containing 2% tryptone, 1% yeast extract material, 2% glucose, 1M sorbitol), and cultured...

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Abstract

The invention belongs to the technical field of gene engineering and enzyme engineering, specifically to a feruloyl esterase A mutant and the purpose thereof. According to the invention, parent genes of feruloyl esterase A undergo a random mutation by error-prone PCR to construct a random mutation library; 12 heat-resistance enhanced feruloyl esterase A mutant sites are selected by a high-throughput screening system; and a complete sequence synthetic method is adopted to integrate the 12 mutation sites onto a coding sequence so as to obtain a combination mutant. The combination mutant has much more enhanced heat stability than the parents and is of potential application value in industrial fields such as bioenergy, forage, papermaking, health food, medicine and the like.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and enzyme engineering, and specifically relates to a ferulic acid esterase A mutant and its application. Background technique [0002] Ferulic acid esterase (EC 3.1.1.73) is able to hydrolyze the ester bonds between hydroxycinnamic acid compounds, arabinoxylan and some gums present in the plant cell wall, and is required for the complete degradation of hemicellulose one of the key enzymes. Among them, feruloyl esterase A from Aspergillus niger is one of the most studied feruloyl esterases. People have done research on its scope of substrate action, endogenous expression regulation, and expression in Pichia pastoris and Escherichia coli. Lots of research. Ferulic acid esterase A has great application potential in bioenergy, feed, papermaking, health food, medicine and other industries. In industrial applications of enzymes, thermostability is an important parameter. The reaction o...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/81C12R1/84C12R1/685
Inventor 吴中柳张帅兵裴小琼刘艳
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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