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Salt-tolerant xylosidase mutant t329e and its preparation method and use

A technology of xylosidase and wild xylosidase, which is applied in the field of salt-tolerant xylosidase mutant T329E and its preparation, can solve problems such as lack of catalytic activity, and achieve the effect of enhanced stability

Active Publication Date: 2022-03-22
YUNNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The object of the present invention is to provide a salt-tolerant xylosidase mutant T329E and its preparation method and application. Good enzyme activity after being treated with high concentration of salt

Method used

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  • Salt-tolerant xylosidase mutant t329e and its preparation method and use
  • Salt-tolerant xylosidase mutant t329e and its preparation method and use
  • Salt-tolerant xylosidase mutant t329e and its preparation method and use

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0031] Construction and Transformation of Experimental Example 1 Expression Vector

[0032] According to the xylosidase nucleotide sequence KY391885 (SEQ ID NO.4) recorded in GenBank, the gene hJ14GH43 encoding the wild xylosidase HJ14GH43 was synthesized; the gene t329e (SEQ ID NO.2) encoding the mutant enzyme T329E was also synthesized.

[0033] The synthetic xylosidase nucleotide sequence and mutant enzyme T329E nucleotide sequence were respectively connected with the expression vector pEasy-E1 to obtain the expression vector containing hJ14GH43 and t329e, and the connection products were respectively transformed into Escherichia coli BL21 (DE3) to obtain the respective Recombinant strain expressing wild enzyme HJ14GH43 and mutant enzyme T329E.

experiment example 2

[0034] Preparation of Experimental Example 2 Wild Enzyme HJ14GH43 and Mutant Enzyme T329E

[0035] The recombinant strains containing hJ14GH43 and t329e were inoculated in LB (containing 100 μg mL - 1 Amp) medium, shake rapidly at 37°C for 16h.

[0036] Then, the activated bacterial solution was inoculated into fresh LB (containing 100 μg mL -1 Amp) culture medium, rapid shaking culture for about 2 ~ 3h (OD 600 After reaching 0.6-1.0), add IPTG at a final concentration of 0.1 mM for induction, and continue shaking culture at 20° C. for about 20 h.

[0037] Centrifuge at 12000rpm for 5min to collect the bacteria. After suspending the cells with an appropriate amount of pH7.0 Tris-HCl buffer solution, the cells were ultrasonically disrupted in a low-temperature water bath.

[0038] After the crude enzyme solution concentrated in the cells was centrifuged at 12,000rpm for 10min, the supernatant was aspirated and the target protein was affinity and eluted with Nickel-NTAAgaro...

experiment example 3

[0040] Determination of the properties of wild enzyme HJ14GH43 and mutant enzyme T329E purified in Experimental Example 3

[0041] The activities of the purified wild enzyme HJ14GH43 and the mutant enzyme T329E were determined by the pNP method, as follows:

[0042] Dissolve pNPX in the buffer solution to make the final concentration 2mM; the reaction system contains 50μL of appropriate enzyme solution and 450μL of 2mM substrate; after the substrate is preheated at the reaction temperature for 5min, add the enzyme solution and react for an appropriate time, then add 2mL 1M Na 2 CO 3 The reaction was terminated, and the released pNP was measured at a wavelength of 405 nm after cooling to room temperature; 1 enzyme activity unit (U) was defined as the amount of enzyme required to decompose the substrate to produce 1 μmol pNP per minute.

[0043] 1. Stability of purified wild enzyme HJ14GH43 and mutant enzyme T329E in NaCl

[0044] The purified enzyme solution was placed in 3....

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Abstract

The invention discloses a salt-tolerant xylosidase mutant T329E and its preparation method and application. The amino acid sequence of the mutant T329E is obtained by mutating the threonine at position 329 of wild xylosidase HJ14GH43 into glutamic acid. Its sequence is shown in SEQ ID NO.1, and the salt is not NaCl. Compared with the wild enzyme HJ14GH43, the mutant enzyme T329E of the present invention has a high concentration of KCl, Na 2 SO 4 and (NH 4 ) 2 SO 4 The stability in the medium has been enhanced, after 15.0-30.0% concentration of KCl treatment, its activity remains 59-70%; after 15.0-30.0% concentration of Na 2 SO 4 After treatment, its activity remained 78-87%; after 20.0-30.0% concentration (NH 4 ) 2 SO 4 After treatment, its activity is increased by about 20%, which can be applied to industries such as tanning, papermaking, and sewage treatment.

Description

technical field [0001] The invention relates to a xylosidase mutant, in particular to a salt-tolerant xylosidase mutant T329E and its preparation method and application. Background technique [0002] Xylan is mainly derived from plant cell walls, accounting for about 15% to 35% of the dry weight of plant cells. Its main chain is polymerized by xylose and has various side chain substituent groups. Endo-xylanase (endo-1,4-β-D-xylanase, EC3.2.1.8) can randomly cut the backbone of xylan to generate xylooligosaccharides, while xylosidase (β- D-xylosidase, EC 3.2.1.37) can hydrolyze xylooligosaccharides into xylose (Collins et al. FEMS Microbiology Reviews, 2005, 29:3-23.). Xylose can be used as a raw material for the production of ethanol, lactic acid, xylitol, etc. In addition to xylan, plant glycoproteins and animal proteoglycans also contain xylose, which can be degraded by xylosidase (Leszczuk et al. Plant Physiology and Biochemistry, 2019, 139:681~690; Takagaki et al. al....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/70C12N1/21C02F3/34C12R1/19C02F103/24C02F103/28
CPCC12N9/2402C12N15/70C12Y302/01037C02F3/342C02F2103/24C02F2103/28
Inventor 周峻沛黄遵锡张蕊李娜韩楠玉唐湘华
Owner YUNNAN NORMAL UNIV
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