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Salt-tolerant xylosidase mutant K321D as well as preparation method and application thereof

A technology of xylosidase and wild xylosidase, which is applied in the field of salt-tolerant xylosidase mutant K321D and its preparation, and can solve problems such as lack of stability

Active Publication Date: 2020-03-24
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 K321D and its preparation method and application. Good enzyme activity after being treated with high salt concentration

Method used

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  • Salt-tolerant xylosidase mutant K321D as well as preparation method and application thereof
  • Salt-tolerant xylosidase mutant K321D as well as preparation method and application thereof
  • Salt-tolerant xylosidase mutant K321D as well as preparation method and application thereof

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 coding gene hJ14GH43 of the wild xylosidase HJ14GH43 was synthesized; the coding gene k321d (SEQ ID NO.2) of the mutant enzyme K321D was synthesized;

[0033] The synthetic coding gene hJ14GH43 and coding gene k321d sequences were respectively connected with the expression vector pEasy-E1 to obtain the expression vector containing hJ14GH43 and k321d, and the ligation products were respectively transformed into Escherichia coli BL21 (DE3), and the wild enzymes HJ14GH43 and Recombinant strain of mutant enzyme K321D.

Embodiment 2

[0034] Embodiment 2 Preparation of wild enzyme HJ14GH43 and mutant enzyme K321D

[0035] The recombinant strains containing hJ14GH43 and k321d 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 (isopropyl-β-D-thiogalactopyranoside) with a final concentration of 0.1 mM for induction, and continue shaking culture at 20° C. for about 20 h.

[0037] Centrifuge at 12,000 rpm for 5 minutes to collect the bacteria, suspend the bacteria with an appropriate amount of Tris-HCl buffer solution with a pH of 7.0, and then ultrasonically disrupt the bacteria 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...

Embodiment 3

[0040] The property determination of the wild enzyme HJ14GH43 of embodiment 3 purification and mutant enzyme K321D

[0041] The activities of the purified wild enzyme HJ14GH43 and the mutant enzyme K321D were determined by the pNP method, as follows:

[0042] Dissolve pNPX in buffer so that the final concentration is 2mM; the reaction system contains 50μL of appropriate enzyme solution and 450μL of 2mM substrate; 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 K321D in NaCl

[0044] The purified enzyme solution was placed in 3.0-30.0% (w / v) NaCl aqueous solution, treated at 20°C for 60 minutes, and then the enzymatic reaction was carried out at pH 7.0 and 20°C, and the untreated e...

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Abstract

The invention discloses salt-tolerant xylosidase mutant K321D as well as a preparation method and application thereof. The amino acid sequence of the mutant K321D is obtained by mutating lysine at the321st site of wild xylosidase HJ14GH43 into aspartic acid, the sequence of the mutant K321D is shown as SEQ ID NO.1, and the salt is not NaCl. Compared with wild enzyme HJ14GH43, the mutant has the advantages that the stability of the mutant enzyme K321D disclosed by the invention in high-concentration KCl, Na2SO4 and (NH4) 2SO4 is enhanced; the activity increased from 48% to 65% after being treated with KCl with the concentration of 20.0%-30.0%, and the activity is 93%-111% after being treated with Na2SO4 with the concentration of 10.0%-30.0%; and the activity is maintained to be higher than90% after being treated with (NH4) 2SO4 with the concentration of 20.0-30.0%; and the salt-tolerant xylosidase mutant K321D can be applied to industries such as tanning, papermaking, sewage treatmentand the like.

Description

technical field [0001] The invention relates to a xylosidase mutant, in particular to a salt-tolerant xylosidase mutant K321D and its preparation method and use. 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. The Jou...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/56C12N15/70C12N1/21C12R1/19
CPCC12N9/2402C12N15/70C12Y302/01037
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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