Acetylxylan esterase and application thereof

An acetylxylan ester and deacylating technology, applied in the field of acetylxylan esterase and its application, can solve the problems of poor activity and performance, and limit wide application

Active Publication Date: 2013-03-06
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, except the acetylxylan esterase of Bacillus subtilis ATCC6633 which was reported to have higher pNPA enzyme activity (1580 U/mg), the activity and performance of the ac

Method used

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  • Acetylxylan esterase and application thereof
  • Acetylxylan esterase and application thereof
  • Acetylxylan esterase and application thereof

Examples

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Embodiment 1

[0045] This embodiment illustrates the genetically engineered bacteria Escherichia coli ( Escherichia coli ) BL21 - Construction of pET-Cah.

[0046] The genetically engineered bacterium Escherichia coli ( Escherichia coli ) BL21 - pET-Cah, which is the Bacillus subtilis ( Bacillus subtilis ) The gene Cah of acetylxylan esterase from CICC 20034 was transformed by the expression vector pET22b E. coli Genetically engineered bacteria obtained from BL21 (DE3).

[0047] The construction method of the above-mentioned genetically engineered bacteria is as follows: using PCR technology to amplify the Cah gene from the plasmid vector Bacillus subtilis genomic DNA, connecting the Cah gene to the multiple cloning site of the expression vector pET22b to obtain the recombinant plasmid pET-Cah, and then Transformation of recombinant plasmid pET-Cah E. coli BL21 (DE3), the recombinant Escherichia coli, that is, the genetically engineered bacteria that produces acetyl xylan esteras...

Embodiment 2

[0058] This example illustrates the induction expression method of acetyl xylan esterase.

[0059] The specific scheme of utilizing the genetically engineered bacterium of embodiment 1 to induce expression of acetyl xylan esterase is as follows:

[0060] (1) Starting strain: Escherichia coli ( Escherichia coli ) BL21 - pET-Cah;

[0061](2) Seed culture:

[0062] Medium: LB medium: yeast powder 5g / L, peptone 10 g / L, sodium chloride 10 g / L;

[0063] Culture conditions: 250 mL Erlenmeyer flask, liquid volume 50 mL, culture temperature 37 ℃, shaker speed 200 r / min, culture 12 h;

[0064] (3) Fermentation culture:

[0065] Medium composition: LB medium: yeast powder 5g / L, peptone 10 g / L, sodium chloride 10 g / L;

[0066] Culture conditions: inoculum size 1.5% (v / v), fermentation temperature 37 ℃, add isopropyl-β-D-thiogalactopyranoside (IPTG) induction when OD reaches 0.6~0.8, the final concentration of IPTG 1.0 mmol / L, shaker speed 180~250 r / min, fermentation 2-10 h.

[00...

Embodiment 3

[0080] This example illustrates the purification, molecular weight and multimeric form of acetyl xylan esterase.

[0081] 1. Purification of acetyl xylan esterase:

[0082] Acetyl xylan esterase was concentrated and purified by 50%-80% (w / v) ammonium sulfate fractional precipitation and ultrafiltration. The purification steps are as follows:

[0083] (1) 50%-80% ammonium sulfate fractional precipitation: take the broken solution, centrifuge at 12000g for 30min; transfer 50mL supernatant to a 100mL beaker, slowly add 50mL of SAS solution (saturated ammonium sulfate solution) while stirring, After the solution was stirred on a magnetic stirrer for 6 hours, it was placed in a 4-degree refrigerator overnight to fully precipitate. The protein solution was centrifuged at 10000 g for 30 min. Transfer 50mL of the supernatant to a new beaker, slowly add 75mL of SAS solution while stirring, so that the final concentration is 80% ammonium sulfate solution, put the solution on a magnet...

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Abstract

The invention discloses DNA (Deoxyribose Nucleic Acid) of a novel acetylxylan esterase, which has a nucleotide sequence shown as SEQ AXENO.1 and an amino acid sequence shown as SEQAXENO.2. Highest activity is obtained when a protein structure is a homotrimer. The invention also discloses a cloning vector containing the DNA of the novel acetylxylan esterase, a cloning technology and application of a recombinant esterase to hydrolysis of a short-chain fatty acid and acyl saccharides and application of the recombinant esterase to production of deacetylated 7-aminocephalosporanic acid and deacetylated cephalosporin C which are important intermediates of beta-lactam antibiotics; and the novel acetylxylan esterase has an important industrial application value.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a Bacillus subtilis ) obtained in the novel esterase and expression vector, recombinant esterase and recombinant esterase in the hydrolysis of short-chain fatty acid lipids, deacylation reaction and important intermediates of β-lactam antibiotics - deacylated 7-aminocephalosporin Acid and deacylated cephalosporin C application. Background technique [0002] Acetyl xylan esterase (AXE for short) is a class of esterases that can deacylate the acylated xylopyranose in xylan or xylooligosaccharides and release acetic acid, classified in carboxyl Esterase family 7, which can hydrolyze short-chain fatty acids and various acylated compounds, has broad substrate specificity. [0003] Nowadays, acetyl xylan esterase has shown great scientific research value and industrial application value, which are mainly reflected in the following two aspects. On the one hand, acetylxyl...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/63C12P7/40C12P35/00C12P35/06
Inventor 黄和李霜田倩倩宋萍徐晴
Owner NANJING UNIV OF TECH
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