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Ammonium sulfate-resistant xylosidase mutant V322DH328DT329E

A technology of V322DH328DT329E and xylosidase, applied in the field of genetic engineering, can solve the problems of simultaneous application, unfavorable degradation of xylo-oligosaccharides, no catalytic activity, etc., and achieves the effect of enhanced stability

Active Publication Date: 2020-04-14
YUNNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Salt-intolerant xylosidase will not be able to be applied simultaneously with chemical fertilizers, which is not conducive to the degradation of xylo-oligosaccharides in agricultural waste, resulting in reduced recycling of xylan and further reduced soil fertility
Due to the salting-out effect at high salt concentration, most enzymes do not have good catalytic activity at high salt concentration

Method used

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  • Ammonium sulfate-resistant xylosidase mutant V322DH328DT329E
  • Ammonium sulfate-resistant xylosidase mutant V322DH328DT329E
  • Ammonium sulfate-resistant xylosidase mutant V322DH328DT329E

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Construction and transformation of embodiment 1 expression vector

[0033] 1) 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 v322d of the mutant enzyme V322D (SEQ ID NO.5) was synthesized ( SEQ ID NO.6) and the coding gene v322dh328dt329e (SEQ ID NO.2) of the mutant enzyme V322DH328DT329E;

[0034] 2) Link the sequences synthesized in (1) with the expression vector pEasy-E1 to obtain the expression vectors containing hJ14GH43, v322d and v322dh328dt329e respectively;

[0035] 3) The ligation products were transformed into Escherichia coli BL21(DE3) to obtain recombinant strains expressing wild enzyme HJ14GH43, mutant enzymes V322D and V322DH328DT329E respectively.

Embodiment 2

[0036] Example 2 Preparation of wild enzyme HJ14GH43 and mutant enzymes V322D and V322DH328DT329E

[0037] The recombinant strains containing hJ14GH43, v322d and v322dh328dt329e were inoculated in LB (containing 100 μg mL -1 Amp) medium, shake rapidly at 37°C for 16h.

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

Embodiment 3

[0040] Example 3 Determination of the properties of the purified wild enzyme HJ14GH43 and mutant enzymes V322D and V322DH328DT329E

[0041] The activities of the purified wild enzyme HJ14GH43 and mutant enzymes V322D and V322DH328DT329E were measured by the pNP method: pNPX was dissolved in buffer to make the final concentration 2mM; the reaction system contained 50μL of appropriate enzyme solution and 450μL of 2mM substrate; After preheating at the reaction temperature for 5 minutes, add the enzyme solution and react for an appropriate time, then add 2mL of 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.

[0042] 1) Stability of purified wild enzyme HJ14GH43 and mutant enzymes V322D and V322DH328DT329E in KCl

[0043] The purified enzyme solution was place...

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Abstract

The invention relates to the technical field of gene engineering and protein modification, and discloses an ammonium sulfate-resistant xylosidase mutant V322DH328DT329E. The amino acid sequence of themutant V322DH328DT329E is as shown in SEQ ID NO.1. After the V322DH328DT329E is treated by 15.0-30.0% (w / v) of KCl, Na2SO4 and (NH4)2SO4 for 60 minutes, the activity of the V322DH328DT329E is 63-85%,60-81% and 99-107% respectively. The ammonium sulfate-resistant xylosidase mutant V322DH328DT329E disclosed by the invention can be applied to the industries of agriculture, tanning, sewage treatmentand the like.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and relates to protein modification technology, in particular to an ammonium sulfate-resistant xylosidase mutant V322DH328DT329E. Background technique [0002] Xylan is a hemicellulose widely present in the cell walls of plants, accounting for about 20% of the dry weight in higher plants and agricultural wastes. Endo-xylanase (endo-1,4-β-D-xyla nase, EC 3.2.1.8) acts on the backbone of xylan, which can randomly cut xylan to generate xylooligosaccharides, xylan Glycosidase (β-D-xylosidase, EC3.2.1.37) can hydrolyze xylooligosaccharides into xylose (Collins et al. FEMS Microbiology Reviews, 2005, 29:3~23.), and xylose can be used as microorganisms, etc. A carbon source for biological utilization, or as a raw material for the production of ethanol, lactic acid, xylitol, etc. [0003] In addition to xylan, plant glycoproteins also contain xylose, which can be degraded by xylosidase (Lesz...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/56C12N15/70C12N1/21C02F3/34C12R1/19
CPCC02F3/342C12N9/2402C12N15/70C12Y302/01037
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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