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Mutant gene TlXynA_2 of xylanase TlXynA and application thereof

A technology of xylanase and mutant gene, which is applied in the field of genetic engineering to achieve the effects of increasing digestion and absorption, a simple and practical method, and reducing viscosity

Active Publication Date: 2016-08-24
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the practical application of TlXynA, there are still problems to be solved
For example, many steps in industrial production are often carried out in the environment of high salt concentration and presence of trypsin, which often limits the high enzyme activity of TlXynA under optimal conditions.
In order to expand the industrial application of TlXynA, it is a very critical task to improve its properties such as high temperature resistance, salt resistance, and trypsin resistance. Reports on its related mutant genes

Method used

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  • Mutant gene TlXynA_2 of xylanase TlXynA and application thereof
  • Mutant gene TlXynA_2 of xylanase TlXynA and application thereof
  • Mutant gene TlXynA_2 of xylanase TlXynA and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Construct the recombinant vector containing the mutant gene TlXynA_2 of xylanase TlXynA, the specific steps are as follows:

[0025] (1) Obtain the xylanase TlXynA gene from the NCBI database, connect the TlXynA gene and the plasmid pET28a to obtain the pET28a-TlXynA plasmid, and its nucleotide sequence is shown in SEQ ID NO.3. Using the above-mentioned recombinant plasmid as a template, design mutation primers for site-directed mutation. The primer sequences are as follows:

[0026]

[0027] The PCR amplification reaction system is as follows:

[0028]

[0029] The reaction procedure is as follows:

[0030]

[0031]

[0032] (2) Take 3 μL of the PCR product for agarose gel electrophoresis verification, leaving a reaction product with clear bands. The wild-type plasmid in the product was digested with DpnI restriction enzyme to ensure that the transformants in the subsequent steps were all mutants. The digestion system is as shown in the table below, mix ...

Embodiment 2

[0041] Construct the recombinant engineered bacterium that contains above-mentioned mutant gene TlXynA_2, concrete steps are as follows:

[0042] Add 2 μL of the above mutant plasmid with correct sequencing to 50 μL E. coli BL-21 competent cells, ice bath for 30 min; Centrifuge for 2 minutes, discard the supernatant (leave a little bottom liquid). Spread the remaining solution on an LB plate containing 50 μg / mL kanamycin, spread evenly until dry, and incubate overnight at 37°C; pick a single clone the next day, and inoculate it in 5 mL of LB medium containing antibiotics, at 37°C Cultivate overnight at 200rpm to obtain recombinant engineered bacteria containing the mutant gene TlXynA_2.

Embodiment 3

[0044] Fermentative expression and purification of the mutant xylanase TlXynA-R116QR161QR187S encoded by the above mutant gene TlXynA_2, the specific steps are as follows:

[0045] (1) Heterologous expression of recombinant protein:

[0046] 1. Get the recombinant engineered bacterium containing the above-mentioned mutant gene TlXynA_2 obtained in Example 2, and cultivate overnight at 37° C. 200 rpm in 5 mL LB medium (containing 50 μg / mL kanamycin);

[0047] ②Transfer the cultured bacterial solution overnight to a 1L Erlenmeyer flask filled with 300mL LB medium (containing 50μg / mL kanamycin), and incubate at 37°C for about 3h until OD 600 =0.6-0.8;

[0048] ③Add IPTG with a final concentration of 0.5mM, and induce culture at 20°C for 20h;

[0049] ④ Centrifuge at 8000rpm at 4°C for 10min to obtain bacterial pellet;

[0050] ⑤NaH with pH 8.0 2 PO 4 -Resuspend the bacteria in NaCl buffer, and place the bacteria in a 100mL centrifuge tube;

[0051] ⑥Put the centrifuge tube ...

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Abstract

The invention discloses a mutant gene TlXynA_2 of xylanase TlXynA. The mutant gene is characterized in that the mutation sites of the gene are R116Q, R161Q and R187S, and a nucleotide sequence of the gene is shown in SEQ ID NO.1. The invention also discloses an application of the gene to preparation of xylanase. Experiments prove that the activity of mutant xylanase coded by the gene can be 3623IU / mg and is 99.7% of the activities of wild type enzymes; and when the external-added salt concentration is 5M, the relative activity of mutant xylanase is 1.50 times the activities of the wild type enzymes. Mutant xylanase is predicted to have the characteristic of high temperature and salt resistance and have broad application prospects in industrial production of feed, food, papermaking, medicine, energy, and the like.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a mutant gene TlXynA_2 of xylanase TlXynA and an application thereof. Background technique [0002] Xylan is the most abundant plant cell wall polysaccharide after cellulose, accounting for 1 / 3 of the total biomass resources on the earth. Its main chain skeleton is composed of xylose connected by β-1,4 glycosidic bonds, and the side chains include various functional groups, such as glucuronic acid, ferulic acid, arabinose, acetyl group, etc. This complex structure leads to the degradation of xylan requires the joint action of a variety of enzymes. The key enzyme is endo-β-1,4-xylanase, which can degrade the main chain by hydrolyzing β-1,4-glycosidic bonds, and convert xylan into xylooligosaccharides. In 2008, Xylooligosaccharide was approved by the Chinese Ministry of Health as a new functional food. It is safe, efficient and stable, and has broad prospects in the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/42C12N15/70
CPCC12N9/2482C12Y302/01008
Inventor 王禄山刘诗佳吴秀芸张怀强
Owner SHANDONG UNIV
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