A kind of xylosidase mutant h328d resistant to sodium chloride and potassium chloride and its application

A technology of xylosidase and mutants, which is applied in the field of genetic engineering, can solve problems such as the influence of enzyme properties, simultaneous application and simultaneous use of chemical fertilizers, and achieve the effect of enhanced stability

Active Publication Date: 2021-06-04
YUNNAN NORMAL UNIV
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, salt can have a dramatic effect on the properties of enzymes
In neutral salts, enzymes will undergo salting-out, resulting in most enzymes not having good catalytic activity at high salt concentrations
Salt-intolerant xylosidases cannot be used with sodium chloride at the same time, which is not conducive to the improvement of pickled food; salt-intolerant xylosidases cannot be used with chemical fertilizers at the same time, which is not conducive to the degradation of xylooligosaccharides in agricultural waste. As a result, the recycling of xylan is reduced, which further leads to the reduction of soil fertility.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A kind of xylosidase mutant h328d resistant to sodium chloride and potassium chloride and its application
  • A kind of xylosidase mutant h328d resistant to sodium chloride and potassium chloride and its application
  • A kind of xylosidase mutant h328d resistant to sodium chloride and potassium chloride and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Construction and transformation of embodiment 1 expression vector

[0032] 1) 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 h328d (SEQ ID NO.2) encoding the mutant enzyme H328D was synthesized;

[0033] 2) Link the sequences synthesized in (1) with the expression vector pEasy-E1 to obtain the expression vectors containing hJ14GH43 and h328d respectively;

[0034] 3) The ligation products were transformed into Escherichia coli BL21(DE3) to obtain recombinant strains expressing the wild enzyme HJ14GH43 and the mutant enzyme H328D respectively.

Embodiment 2

[0035] Embodiment 2 Preparation of wild enzyme HJ14GH43 and mutant enzyme H328D

[0036] The recombinant strains containing genes hJ14GH43 and h328d were inoculated in LB (containing 100 μg mL -1 Amp) medium, shake rapidly at 37°C for 16h.

[0037] 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 with 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 and 0-500 mM imidazole, res...

Embodiment 3

[0039] The property determination of the wild enzyme HJ14GH43 of embodiment 3 purification and mutant enzyme H328D

[0040] The activity of purified wild enzyme HJ14GH43 and mutant enzyme H328D was measured by pNP method: Dissolve pNPX in buffer to make the final concentration 2mM; After preheating for 5 minutes, add 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.

[0041] 1) Stability of purified wild enzyme HJ14GH43 and mutant enzyme H328D in NaCl

[0042] 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 enzyme solution was used as a control. Us...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to the technical field of genetic engineering and protein transformation, and discloses a sodium chloride- and potassium chloride-resistant xylosidase mutant H328D and its application. The amino acid sequence of the mutant H328D is shown in SEQ ID NO.1. The mutant has good stability in high concentrations of NaCl and KCl. After being treated with 3.0-30.0% (w / v) NaCl for 60 minutes, the activity is 44-60%; After 60 minutes of treatment, the activity was 73-105%. The sodium chloride and potassium chloride resistant xylosidase mutant H328D of the present invention can be applied to industries such as food, agriculture, and sewage treatment.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and relates to protein modification technology, in particular to a sodium chloride and potassium chloride resistant xylosidase mutant H328D and application thereof. Background technique [0002] Xylan is the main component of plant hemicellulose, widely exists in plant cell walls, and accounts for about 20% of dry weight in higher plants and agricultural waste. Endo-xylanase (endo-1,4-β-D-xylanase, EC 3.2.1.8) can randomly cut the backbone of xylan to generate xylooligosaccharides, xylosidase (β-D- xylosidase, EC 3.2.1.37) can hydrolyze xylooligosaccharides into xylose (Collins et al.FEMS Microbiology Reviews, 2005, 29:3~23.), and xylose can be used as a carbon source for microorganisms and other organisms, or as a Raw materials for the production of ethanol, lactic acid, xylitol, etc. [0003] Salt widely exists in nature and in various production practices, including food, agricult...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N1/21A23L29/00C02F3/34C12R1/19
CPCA23V2002/00A23L29/06C02F3/342C12N9/248C12N15/70C12Y302/01037A23V2250/54
Inventor 周峻沛黄遵锡张蕊李娜韩楠玉唐湘华
Owner YUNNAN NORMAL UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap