Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Recombinant basic xylanase resistant to chelating agent ethylenediamine tetraacetic acid and construction method thereof

A xylanase and alkaline technology, which is applied in the field of industrial enzyme development and utilization, can solve the problems of enzyme activity reduction and achieve the effects of saving process water, reducing use, and huge ecological benefits

Active Publication Date: 2015-09-23
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
View PDF3 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The mature fragment xynA-M of the gene was expressed in Escherichia coli, and the obtained mature xylanase was purified, and the study of enzymatic properties showed that this alkaline xylanase was stable at pH 9.2, 60°C and 5mM EDTA conditions, the enzyme activity was significantly reduced

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
  • Recombinant basic xylanase resistant to chelating agent ethylenediamine tetraacetic acid and construction method thereof
  • Recombinant basic xylanase resistant to chelating agent ethylenediamine tetraacetic acid and construction method thereof
  • Recombinant basic xylanase resistant to chelating agent ethylenediamine tetraacetic acid and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] The invention discloses a group of EDTA-resistant alkaline xylanase and its encoding gene, which is characterized in that the alkaline xylanase gene xynA contained in GenBank accession number EU421717.1 corresponds to the amino acid sequence of xylanase xynA The accession number is ACA00160.1, except the 27 amino acid signal peptide of the N-segment, the remaining 201 amino acids are the mature fragment XynA-M of the xylanase, and the corresponding mature fragment of the xylanase gene is xynA-M . For xynA-M, design point mutation primers (FP1, RP1, FP2, RP2, FP3, RP3) for site-directed mutagenesis, and change the guanine G at positions 56 and 57 of the published gene into adenine A and thymine T, respectively , change the guanine G at positions 56 and 57 to thymine T and cytosine C respectively, and change the thymine T and guanine G at positions 55, 56 and 57 to cytosine C, adenine A and cytosine C respectively , so that tryptophan W (TGG) at the 19th position of the ...

Embodiment 2

[0071] Medium: LB resistant medium: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, kanamycin 50μg / mL;

[0072]Culture method: the recombinant strains BL21(DE3) / pET28a-xynA-M-W19Y, BL21(DE3) / pET28a-xynA-M-W19F and BL21(DE3) / pET28a-xynA-M-W19H in Example 1 were grown in LB After activating and preparing seeds on the resistant plate, transfer them to liquid LB medium at an inoculum size of 1:100, and cultivate to OD at 37°C and 200rpm. 600 If the concentration is 0.6-0.8, add the inducer isopropylthiogalactoside (IPTG) to a final concentration of 0.5mM, and then culture at 18°C ​​and 200rpm for 8 hours.

[0073] The induced bacterial cells were ultrasonically destructed, passed through a HIS column for purification and concentration, and purified xylanase mutants XynA-M-W19Y, XynA-M-W19F and XynA-M-W19H were obtained.

Embodiment 3

[0075] Method for measuring xylanase enzyme activity: Take 20 μl of diluted enzyme solution, add 380 μl of 1% xylan (beech xylan, pH9.2 glycine-NaOH buffer solution) solution, mix well, and place at 60°C for 10 minutes. Immediately add 600 μl 3,5-dinitrosalicylic acid (DNS), boil for 7 minutes, measure OD 540 nm value. The blank control is the first inactivated enzyme solution (that is, first take 20 μl of the enzyme solution and inactivate it in a water bath at 100°C for 5 minutes, then add the substrate to react for 10 minutes). The enzyme activity unit (U) was defined as the amount of enzyme needed to catalyze the production of 1 μmol xylose per minute.

[0076] Enzyme activity calculation formula: A=(M*V1*C) / (V2*T)

[0077] In the formula: A——enzyme activity (U / ml)

[0078] M——enzyme solution dilution multiple

[0079] V1——enzyme reaction volume (ml)

[0080] C——xylose concentration (μmol / ml), which can be calculated according to xylose standard curve and measured OD5...

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 discloses recombinant basic xylanase resistant to chelating agent ethylenediamine tetraacetic acid and a construction method thereof. A basic xylanase encoding gene xynA is mutated by adopting a site-directed mutation technology to obtain mutant genes xynA-M-W19Y, xynA-M-W19F and xynA-M-W19H, induction, expression and purification are performed in E.coli BL21(DE3), enzyme activity determination is performed to purified enzyme, and the obtained xylanase mutants XynA-M-W19Y, XynA-M-W19F and XynA-M-W19H have a better capacity of resisting EDTA, wherein under conditions of pH 9.2, 60DEG C and 5mM EDTA, the enzyme activity of a mutant W20Y is approximate 20 percent higher than the enzyme activity of wild type XynA. Thereby, a good foundation is laid for the wide application of basic xylanase.

Description

technical field [0001] The invention belongs to the field of development and utilization of industrial enzymes, in particular to a group of recombinant alkaline xylanases resistant to chelating agent ethylenediaminetetraacetic acid (EDTA), encoding genes thereof, and a construction method thereof. technical background [0002] The paper industry is one of the six most polluting industries in the world. The annual wastewater discharge of my country's paper industry reaches 400,000 tons, accounting for 1 / 6 of the national industrial wastewater discharge. Over the years, people have made unremitting efforts to try to develop a new technology of pulping and papermaking with no pollution and high efficiency, so as to reduce pollution and protect the environment. The pulping and bleaching processes in the papermaking process are the main processes for the generation of pollutants. Biotechnology can help solve these problems, the most attractive and challenging of which are biopul...

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
IPC IPC(8): C12N9/42C12N15/56C12N15/70
Inventor 马延和邢霞王钦宏涂然彭彦峰
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com