Xylanase PaXynA with low-temperature activity and coding gene and application thereof

A technology of xylanase activity and xylan, which is applied in the biological field, can solve problems such as poor low temperature adaptability, and achieve the effects of reducing industrial production costs, saving energy and reducing environmental pollution

Active Publication Date: 2020-12-01
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to overcome the problem of poor low temperature adaptability of existing xylanases, and provide a gene, protein, and method for cracking xylan of xylanase PaXynA and its application

Method used

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  • Xylanase PaXynA with low-temperature activity and coding gene and application thereof
  • Xylanase PaXynA with low-temperature activity and coding gene and application thereof
  • Xylanase PaXynA with low-temperature activity and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 is the construction of the recombinant vector of the Paenibacillus aerobacillus R14 xylanase gene.

[0045] In this embodiment, the Escherichia coli clone strain competent cell DH5α and the expression strain competent cell BL21 (DE3) are commercially available products of Beijing Quanshijin Company;

[0046] Expression vector pET-28a(+): commercially available from Novagen;

[0047] LB medium consists of 1L ddH 2 Prepared by adding 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl to O, and sterilizing at 121°C for 30 minutes;

[0048] 50 mg / mL of kanamycin using 20 mL of sterile ddH 2 Dissolve 1 g of kanamycin in O, prepare a stock solution with a concentration of 50 mg / mL, and store it at -20°C for later use.

[0049] Using Paenibacillus aerobacillus R14 as the original bacteria, expanding the culture and extracting its genomic DNA as an amplification template; the Paenibacillus aerobacillus R14 is a new species of Paenibacillus preserved in the Gu...

Embodiment 2

[0058] Example 2 is the induced expression of the recombinant protein of Paenibacillus aerobacillus R14 xylanase.

[0059] In this example, 1mol / L IPTG (isopropyl-β-D-thiogalactoside) was dissolved in 10mL sterilized ddHO by weighing 2.383g IPTG 2 In O, configure it as a storage solution of IPTG with a concentration of 1mol / L, and store it at -20°C for later use;

[0060] LB culture medium and kanamycin preparation method and composition are as shown in embodiment 1;

[0061] NTA-0 solution was prepared by adding 7.8g NaH 2 PO 4 , 17.5g NaCl dissolved in 1LddH 2 O, obtained by adjusting pH7.5-8.0;

[0062] The imidazole solution of 500mmol / L is made up of 34.04gC 3 h 4 N 2 Dissolved in 1L NTA-0 solution and adjusted to pH=8.0.

[0063] The recombinant vector constructed in Example 1 was transformed into Escherichia coli BL21 (DE3) and activated on an LB plate containing 50 μg / mL kanamycin resistance. Cultured in LB liquid medium, 37°C, 220rpm, cultured for 12h. Measu...

Embodiment 3

[0067] Example 3 is the determination of the enzymatic properties of xylanase PaXynA.

[0068] In the present embodiment, oat xylan is a product sold by Megazyme Company;

[0069] DNS reagent: weigh 50g of 3,5-dinitrosalicylic acid and dissolve it in 4000mL of water, stir constantly, slowly add 80g of NaOH to dissolve it completely, continue stirring, add 1500g of potassium sodium tartrate in small portions, and heat carefully , the maximum temperature of the solution does not exceed 45°C. After cooling to room temperature, make up to 5000mL. If the solution is not clear, filter it with filter paper, then store the brown bottle at room temperature, and use it after one week;

[0070] 1% xylan: add 1g oat xylan to 80mL 0.1mol / L pH7.0 Tris-HCl buffer solution, heat in a boiling water bath for 10min, adjust the pH to 7.0 after cooling, then add buffer solution to make the volume 100mL. Centrifuge at 10,000×g for 5 minutes, take the supernatant, and store it at 4°C for later us...

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Abstract

The invention relates to xylanase PaXynA with low-temperature activity and a coding gene and application thereof. The protein has xylanase activity, and the amino acid sequence of the protein has at least 99% consistency with SEQ ID NO: 2. The xylanase PaXynA disclosed by the invention has good low-temperature adaptability.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a protein of xylanase PaXynA, a gene encoding the protein, an expression vector containing the gene, a recombinant strain, a method for cracking xylan and an application thereof. Background technique [0002] Xylan (Xylan) is the main component of plant hemicellulose, which accounts for about 33% of all renewable organic carbon on the earth. It is a polymer formed by β-1,4-glycosidic bonds, and its content in nature is only Lower than cellulose, it has great potential for industrial utilization. Xylanase can hydrolyze xylan, so that xylan can be used in many industrial productions, and has broad application prospects in many industrial fields, including biobleaching of kraft pulp, degumming of plant fibers, tobacco processing, vegetable oil extraction of textile fibers, bioconversion of agricultural waste, and biofuel production. [0003] The structure of xylan is complex, and comp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N1/21C12P19/02C12P19/14C12R1/19
CPCC12N9/2482C12N15/70C12P19/02C12P19/14
Inventor 林敏张维李江高如雨周正富陆伟柯秀彬
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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