Chemically modified xylanase and preparation method and application thereof

A technology of xylanase and chemical modification, applied in the field of enzyme engineering, which can solve the problems of reducing enzyme activity and achieve the effect of increased affinity and high reaction rate

Pending Publication Date: 2021-11-09
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the chemical modification of enzymes can change some properties of enzymes and improve their use efficiency, the structure of enzyme molecules is often destroyed during the modification process, which reduces the activity of enzymes. Therefore, it is necessary to optimize the chemical modification conditions of enzymes, including modification Time, temperature and pH to reduce loss of enzyme activity

Method used

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  • Chemically modified xylanase and preparation method and application thereof
  • Chemically modified xylanase and preparation method and application thereof
  • Chemically modified xylanase and preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0029] Expression and enzyme activity detection of xylanase described in embodiment 1

[0030] Using XylB derived from Aspergillus niger as a template, a mutant fragment containing EcoRI and SalI was designed and ligated to pET28a(+) by one-step cloning to obtain the target plasmid. The plasmid was transformed into Escherichia coli BL21(DE3), the transformation solution was spread on an LB plate and cultured at 37°C for 12 hours, and the single colony grown was the expression strain. Pick a single colony and inoculate it into 5mL LB medium for 12h at 37°C and 200rpm, and then inoculate it into 200mL LB medium for expansion. When the OD600 of the bacterial solution grows to 0.6-0.8, add 0.1mM IPTG to induce culture for 12h.

[0031] The upstream primer of XylB-F is: 5'-ATGGGTCGCGGATCCGAATTCATGTTTAAATTTA AAAAAAATTTC-3'

[0032] The upstream primer of XylB-R is: 5'-TGCGGCCGCAAGCTTGTCGACCCATACAGTCAC GTTAGAGCTA-3'

[0033] Enzyme activity detection: 0.2mL enzyme solution and 1mL ...

Embodiment 2

[0035] Fermentative production and purification of xylanase described in embodiment 2

[0036] Inoculate 50 μL of the seed-preserving bacteria solution into 5 mL of LB medium for 12 hours at 37°C and 200 rpm, and then inoculate into 200 mL of fermentation medium to expand the culture. When the OD600 of the bacteria solution grows to 0.6-0.8, add 0.1 mM IPTG to induce culture for 12 hours . After the fermentation, the fermentation broth was ultrasonically crushed, centrifuged to take the supernatant after crushing, and ammonium sulfate solution with 100% saturation of the same volume was added to it, stirred at 4°C for 2 hours, then stood still for 6 hours, centrifuged to obtain a precipitate, and added pH 7.0 Preserve the PBS dissolved pellet. The method and condition optimization of xylanase chemical modification described in embodiment 3

Embodiment 3

[0036] Inoculate 50 μL of the seed-preserving bacteria solution into 5 mL of LB medium for 12 hours at 37°C and 200 rpm, and then inoculate into 200 mL of fermentation medium to expand the culture. When the OD600 of the bacteria solution grows to 0.6-0.8, add 0.1 mM IPTG to induce culture for 12 hours . After the fermentation, the fermentation broth was ultrasonically crushed, centrifuged to take the supernatant after crushing, and ammonium sulfate solution with 100% saturation of the same volume was added to it, stirred at 4°C for 2 hours, then stood still for 6 hours, centrifuged to obtain a precipitate, and added pH 7.0 Preserve the PBS dissolved pellet. The method and condition optimization of xylanase chemical modification described in embodiment 3

[0037] Oxidation of dextran: 2g of dextran with a molecular weight of 150kDa and 2.4g of NaIO 4 respectively dissolved in 10mL water, NaIO 4 Pour into the dextran solution, stir and react at 30°C in the dark for 6 hours, t...

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Abstract

The invention discloses chemically modified xylanase and a preparation method and application thereof, and belongs to the field of enzyme engineering. According to the oxidation characteristic of NaIO4, hydroxyl on dextran is oxidized into aldehyde group, and then xylanase is chemically modified. The modified enzyme is of a dextran structure and can be combined with cellulose in lignocellulose to achieve an immobilization effect, so that the enzyme activity is improved by 237%; and meanwhile, the enzyme has a higher maximum reaction rate Vmax. The preparation method overcomes the defect that the reaction rate of xylanase in lignocellulose is low, the catalytic activity of xylanase is improved, and wide industrial application prospects are realized.

Description

technical field [0001] The invention belongs to the field of enzyme engineering, in particular to a chemically modified xylanase and its preparation method and application. Background technique [0002] Xylan is the main component of plant hemicellulose and is the most abundant polysaccharide in nature besides cellulose. The main chain of xylan is a polymer composed of β-D-xylopyranose residues linked by β-1,4-glycosidic bonds. Xylan in plants is located between lignin and cellulose, and there are different degrees of interactions between xylan and other components of lignocellulosic. Xylan mainly connects with lignin through covalent bonds, and interacts with cellulose through non-covalent bonds. One of the difficulties in the degradation of lignocellulose is that it is difficult to degrade due to its complex structure. Therefore, by chemically modifying xylanase, it is linked to dextran so that it can be combined with cellulose to achieve an immobilized effect. , thereb...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/70C12P19/14C12P19/02C12R1/19
CPCC12N9/2482C12Y302/01008C12N15/70C12P19/14C12P19/02C12P2203/00
Inventor 马江锋姜岷王寅竹吴昊董维亮
Owner NANJING UNIV OF TECH
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