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Acetyl xylan esterase gene as well as encoded product and preparation method thereof

A technology for acetyl xylan ester and enzyme gene, which is applied in the field of obtaining acetyl xylan esterase gene and its encoded product, and can solve problems such as insufficient research and pending development.

Active Publication Date: 2020-06-09
GUANGDONG PHARMA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, compared with other lignocellulosic degrading enzymes, the research on acetylxylan esterase is far from enough. The synergy between acetylxylan esterase and other lignocellulose degrading enzymes has been recognized, and its potential application Value is yet to be developed

Method used

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  • Acetyl xylan esterase gene as well as encoded product and preparation method thereof
  • Acetyl xylan esterase gene as well as encoded product and preparation method thereof
  • Acetyl xylan esterase gene as well as encoded product and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Cloning of acetylxylan esterase gene Est1051 and construction of recombinant expression plasmid

[0061] 1. Primer design and synthesis of acetylxylan esterase gene Est1051

[0062] The total DNA was extracted from the soil samples of the wild Ferula ferulae distribution area in Shihezi Nanshan on the southern edge of the Junggar Basin in Xinjiang. The extraction method was:

[0063] (1) Take 12 autoclaved 5mL centrifuge tubes, weigh soil samples, 1.25g in each tube, add 1.5ml of DNA extraction buffer, and activate on a shaker at 130rpm / min at 37°C for 30min.

[0064] (2) Take out the centrifuge tube and add 300 μL of 10% SDS respectively.

[0065] (3) Bathe in a water bath at 65°C for 2 hours, gently invert up and down every 15mm and mix well.

[0066] (4) Take out the sample, after cooling, centrifuge at 10000rpm / min at 4°C for 10min.

[0067] (5) Transfer the supernatant to a new 5mL centrifuge tube, centrifuge at 10000rpm / min at 4°C for 20min.

[0068]...

Embodiment 2

[0112] Example 2 Induced expression optimization and purification of acetylxylan esterase gene Est1051

[0113] The seed solution was prepared according to the following method for later use: select a single colony of the recombinant expression strain and place it in 5 mL of LB liquid medium, and cultivate overnight at 37° C. and 180 rpm for 12 hours with shaking.

[0114] (1) Determination of the optimal induction temperature

[0115] Three different experimental groups were set up according to different induction temperatures. The operation of each test sample in each experimental group was as follows: draw 500 μL of culture solution from the spare seed solution into 50 mL of LB liquid medium, and incubate at 30°C and 220rpm. Shake culture to cell density OD 600nm When it was 1.0, IPTG with a final concentration of 0.1mM was added, and the test samples of each experimental group were placed in a constant temperature shaker at 25°C, 37°C and 40°C, respectively, and induced b...

Embodiment 3

[0126] The enzyme activity assay of embodiment 3 acetyl xylan esterase

[0127] (1) Determination of enzyme activity

[0128] The invention uses p-nitrophenol esters as substrates to detect the enzyme activity of acetyl xylan esterase. The reaction system was 200 μL, which consisted of 10 μL stock solution containing 10 mM p-nitrophenol ester, 180 μL PBS (pH=6.8) buffer solution and 10 μL enzyme solution. The reaction system was reacted at 40° C. for 20 min, and then the absorbance of p-nitrophenol released during the process was measured at a wavelength of 405 nm, and a blank control without enzyme solution was made at the same time. Determine its concentration according to the standard curve of p-nitrophenol. The enzyme activity unit is defined as: under the reaction conditions, the amount of enzyme required to catalyze 1 μmol of p-nitrophenol acetate per minute is defined as one enzyme activity unit.

[0129] (2) Preparation of pNP standard curve

[0130] Prepare a 10 m...

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Abstract

The invention discloses an acetyl xylan esterase gene as well as an encoded product and preparation method thereof. The acetyl xylan esterase gene is named Est1051, and the nucleotide sequence is shown as SEQ ID NO.1; and the amino acid sequence is shown as SEQ ID NO.2. The invention also discloses a recombinant plasmid pET28a-Est1051 containing the acetyl xylan esterase gene, and discloses a preparation method of acetyl xylan esterase. The acetyl xylan esterase gene Est1051 is overexpressed in an escherichia coli prokaryotic expression system, and the acetyl xylan esterase realizes efficientand soluble expression in the escherichia coli expression system; and at the same time, the acetyl xylan esterase obtained by using the method has higher catalytic activity and thermal stability, andhas great industrial production and application prospects.

Description

technical field [0001] The invention relates to the technical field of gene recombination, in particular to the technology of how to obtain the acetyl xylan esterase gene and its encoded product. Background technique [0002] Hemicellulose is the most abundant renewable biological resource besides cellulose in nature. Xylan is an important component of hemicellulose in plant cell walls, accounting for about 15%-35% of the dry weight of plant cells. These xylose polymers are widely connected with groups such as acetyl, arabinose, ferulic acid and 4-O-methyl-D-glucuronic acid residues to form different side chain groups, so xylan has Various structures. The presence of the acetyl group on the side chain of xylan hinders the degradation of xylan by xylanase, which in turn affects the hydrolysis efficiency of the substrate and creates a steric barrier that limits the contact between the main chain degrading enzyme and the main chain. Reduced accessibility. [0003] Acetyl xy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/18C12N15/70C12N1/21C12R1/19
CPCC12N9/18C12N15/70C12Y301/01072Y02E50/10
Inventor 李荷张梦乐
Owner GUANGDONG PHARMA UNIV
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