Neutral high temperature xylanase as well as coding genes and application thereof

A technology encoding gene and xylanase, applied in the field of genetic engineering, can solve the problems of high energy and acid-base chemical reagents, environmental pollution, cost increase, etc.

Active Publication Date: 2017-09-05
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The existing bioethanol processing technology includes acid-base pretreatment of lignocellulose, long-term high-temperature enzyme reactio

Method used

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  • Neutral high temperature xylanase as well as coding genes and application thereof
  • Neutral high temperature xylanase as well as coding genes and application thereof
  • Neutral high temperature xylanase as well as coding genes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Cloning of the genomic DNA of embodiment 1, neutral high-temperature xylanase CtXyn10A

[0074] Extract the genomic DNA of Cladosporium tianshanense SL-14:

[0075] Filter the mycelium cultured in liquid for 3 days with sterile filter paper, put it into a mortar, add 2mL of extract, grind for 5min, then put the grinding solution in a 50mL centrifuge tube, lyse in a water bath at 65°C for 20min, and mix every 10min. Homogenize once and centrifuge at 10,000 rpm for 5 min at 4°C. The supernatant was extracted in phenol / chloroform to remove impurity proteins, and then an equal volume of isopropanol was added to the supernatant. After standing at room temperature for 5 minutes, centrifuge at 10,000 rpm for 10 minutes at 4°C. The supernatant was discarded, the precipitate was washed twice with 70% ethanol, dried in vacuum, dissolved by adding an appropriate amount of TE, and stored at -20°C for later use.

[0076] According to the sequence analysis of the genome framework o...

Embodiment 2

[0083] Example 2, Acquisition of the gene encoding mature neutral high-temperature xylanase CtXyn10A

[0084] Extract the total RNA of Cladosporium tianshanense SL-14, use reverse transcriptase to obtain a strand of cDNA, and then use primers Ctxyn10A-F / Ctxyn10A-R to amplify the single-stranded cDNA, and the amplified product is recovered and sent to III Sequenced by Bo Biotechnology Co., Ltd.

[0085] Ctxyn10A-F: 5'-GGGGAATTCCACCCTTCGGCTCCCAAGGAC-3';

[0086] Ctxyn10A-R: 5'-GGGGCGGCCGCTCACTTCTTGCCACCCTTGATGCC-3';

[0087] After comparing the genome sequence of the nucleic acid fragment measured in Example 1 with the sequencing result of the above-mentioned PCR amplification product, it was found that the gene has 2 introns, the cDNA is 1059bp long, and has the nucleotide of SEQ ID No. 2 in the sequence table Sequence, the amino acid sequence shown in SEQ ID No. 3 in the coding sequence list and a stop codon. A protein with the amino acid sequence shown in SEQ ID №: 3 in th...

Embodiment 3

[0090] Embodiment 3, the preparation of neutral high-temperature xylanase CtXyn10A

[0091] The Pichia pastoris expression vector pPIC9 was subjected to double digestion (EcoRI+NotI), and the nucleic acid fragment encoding the mature protein prepared in Example 2 was double-digested (EcoRI+NotI) to cut out the gene fragment encoding the mature protein and The expression vector pPIC9 after double digestion was ligated.

[0092] Sequencing to verify the correctness of the sequence. The sequence of the foreign gene inserted in the resulting recombinant plasmid is SEQ ID No.: 2 nucleotides 55-1059, and the recombinant plasmid is named pPIC-Ctxyn10A.

[0093] The above-mentioned recombinant plasmid pPIC-Ctxyn10A was transformed into Pichia pastoris GS115; the correctness of the sequence was verified by extracting the plasmid from the positive recombinant bacteria, and the recombinant Pichia strain containing the recombinant plasmid pPIC-Ctxyn10A was named GS115 / Ctxyn10A.

[0094]...

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Abstract

The invention provides a neutral high temperature xylanase CtXyn10A as well as genes and application thereof. The neutral high temperature xylanase CtXyn10A has proteins of amino acid sequences shown in SEQ ID No. 3 and/or SEQ ID No. 5 in a sequence table; and/or is characterized in that amino acid residue sequences shown in SEQ ID No. 3 and/or SEQ ID No. 5 in the sequence table are replaced and/or deleted and/or added by one or several amino acid residues, and have proteins with xylanase activity and derived from the proteins with the amino acid sequences shown in SEQ ID No. 3 and/or SEQ ID No. 5 in the sequence table. The xylanase CtXyn10A has an optimum pH of 6.0 to 7.0 and an optimum temperature of 70 DEG C, keeps enzyme activity of 20% or above at 90 DEG C and pH 9.0, and specific activity is 461 U / mg; the xylanase CtXyn10A has the activity of xylanase, glucanase and cellulase, is easy to produce by industrial fermentation, and can be widely used in food, paper, energy industry and the like as a novel broad-spectrum enzyme preparation.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to a neutral high-temperature xylanase, its encoding gene and its application. Background technique [0002] Xylan (xylan) is the main component of the second most abundant plant biomass in nature after cellulose (Lynd et al. Microbiology and Molecular Biology Review, 2002, 66:506–577), in terms of bioenergy have potential use value. Existing biotechnology can efficiently degrade the cellulose component in plants and convert it into fermentable glucose, which is produced by yeast to produce bioethanol. High-component xylan not only limits the combination reaction of cellulase and cellulose, inhibits the catalytic reaction, but also discharges into nature as a by-product, which can cause nutrient enrichment and damage the ecosystem (Polizeli et al.Applied Microbiology and Biotechnology, 2005, 67:577-591). Therefore, in the process of biomass bioconversion, xylanase is...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/56C12N15/63C12N15/11C12P19/14C12P19/02C12P19/12C12P19/00
CPCC12N9/2482C12P19/00C12P19/02C12P19/12C12P19/14C12Y302/01008
Inventor 姚斌马锐柏映国罗会颖黄火清苏小运王苑涂涛王亚茹孟昆
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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