GH11 family xylanase gene, cloning expression and ramie degumming application thereof

A technology for xylanase and xylan, which is applied in the field of genetic engineering, can solve the problems of poor pH tolerance and low catalytic efficiency of xylanase, and achieves the improvement of both enzyme activities, strong adaptability of metal ions, and significant improvement. The effect of industrial application advantages

Pending Publication Date: 2022-02-01
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention solves the technical problems of low xylanase catalytic efficiency and poor pH tolerance in the prior art, clones and obtains two xylanase genes from Aspergillus terreus, and expresses two xylanases. The xylanase in the present invention is applied to ramie degumming, has high catalytic efficiency, good pH tolerance, wide range of acid and alkali that can be adapted, good thermal stability, and the enzyme has strong adaptability to metal ions and has no Significantly inhibitory ion with good tolerance to most solvents

Method used

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  • GH11 family xylanase gene, cloning expression and ramie degumming application thereof
  • GH11 family xylanase gene, cloning expression and ramie degumming application thereof
  • GH11 family xylanase gene, cloning expression and ramie degumming application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: Gene Cloning of Aspergillus terreus-derived xylanase xyl-1, xyl-2

[0059] Filter the mycelium cultivated for 3-4 days with sterile filter paper and put it into a mortar, add liquid nitrogen to grind, add the extract at the same time, then add 0.2ml chloroform, and centrifuge at 12000rpm for 15min after ice bathing for 10min. Finally, add the centrifuged supernatant to the extraction tube, add RNA wash buffer 1 and 2 respectively for centrifugation, and finally dissolve it in DPEC water, centrifuge to recover the liquid, and store it in a -20°C refrigerator for later use.

[0060] Thermally denature the extracted RNA for 5 minutes, add reaction solution to remove genomic DNA, and perform reverse transcription PCR reaction (reaction at 37°C for 15 minutes, reaction at 50°C for 5 minutes, reaction at 98°C for 5 minutes) to obtain the cDNA sequence, and store at -80°C .

[0061] According to the genome of Aspergillus terreus NIH2624, 2 primers for xylanase xyl-...

Embodiment 2

[0064] Example 2: Heterologous expression of recombinant xylanase xyl-1 and xyl-2

[0065] Inoculate recombinant strains E.coli BL21 / xyl-1 and E.coli BL21 / xyl-2 into 100ml liquid LB culture based on 37°C and 180rpm culture, when OD 6oo When it reaches about 0.6-0.8, add IPTG to induce the expression of the enzyme. Then transfer to 16°C and 100rpm to cultivate for 16h, then centrifuge at 10000rpm for 10min to collect the bacteria. Suspend the cells with an appropriate amount of PBS buffer solution with a pH of 7.0, break the cells with an ultrasonic cell disruptor in an ice bath, centrifuge at 4°C and 8000 rpm / min for 10 min, and the supernatant is the crude enzyme solution.

Embodiment 3

[0066] Embodiment 3: Purification of xylanase xyl-1, xyl-2

[0067] First equilibrate the column with 10 times the column volume Binding buffer, and then load the crude enzyme solution at a flow rate of 20 times the column volume / hour; wash the column with an eluent of 15 times the column volume (10mM imidazole) to wash away the impurity proteins; The target protein was eluted with 5 times column volume Elutionbuffer (200mM imidazole), and the eluate was collected; then the column was washed with 15 times column volume (500mM imidazole) eluent; finally, the purified protein was desalted using a desalting spin column.

[0068] The collected enzyme solution was detected by SDS-PAGE, and the results were as follows: figure 2 shown. The protein concentration was determined with bovine serum albumin (BSA) as the standard.

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Abstract

The invention relates to a GH11 family xylanase gene, cloning expression and ramie degumming application thereof, and belongs to the field of gene engineering. The invention discloses cloning, expression and application of two GH11 family xylanase genes from aspergillus terreus. According to the invention, two GH11 family xylanase genes are excavated from a genome of aspergillus terreus for the first time, and heterologous expression and physicochemical property analysis are carried out; the xylanase disclosed by the invention is high in catalytic efficiency, relatively good in pH tolerance, relatively wide in adaptable acid-base range, relatively good in thermal stability, relatively strong in adaptability to metal ions, free of ions with an obvious inhibition effect and relatively good in tolerance to most solvents; ramie slices are used for catalytic experiment verification, and it is found that the ramie xylan component can be effectively degraded; and the two enzymes have a good degradation effect on ramie xylan components, the tolerance to pH, temperature, metal ions and solvents meets the degumming requirements of ramie, and the two enzymes can be well applied to industrial production.

Description

technical field [0001] The invention relates to the field of genetic engineering, more specifically, relates to GH11 family xylanase gene and its clone expression and ramie degumming application, especially relates to the cloning expression, purification and ramie degumming application of two xylanase genes. Background technique [0002] Hemicellulose accounts for almost one-third of all renewable organic matter on Earth and is the most abundant group of biopolymers after cellulose. Xylan is a hemicellulose widely present in plant cell walls, about Accounting for 20-30%, it is a xylose polymer connected by β-1,4-glycosidic bonds, and its side chain can be replaced by side groups such as acetyl, galacturonic acid, and arabinose. [0003] Xylanase can hydrolyze xylan molecules into xylo-oligosaccharides such as xylose or xylooligosaccharides. Most xylanase families belong to GH10 and GH11 families. The three-dimensional structure of xylan in GH10 family is shown as (a / β ) 8-b...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/70C12N15/10C12N1/21D01C1/00C12R1/19
CPCC12N9/248C12N15/70D01C1/00
Inventor 余龙江吴亚舒潼李攀登王慧慧向梦雄李雁蓉侯梓淇
Owner HUAZHONG UNIV OF SCI & TECH
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