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F52-6 protein and application of coding gene of F52-6 protein and hydrolyzed xylan

A protein and protein technology, applied in the fields of application, hydrolase, genetic engineering, etc., can solve problems that limit the efficient development of the biofuel industry

Active Publication Date: 2015-03-25
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This limits the efficient development of the biofuel industry and is also an important factor restricting the reduction of production costs of biofuels

Method used

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  • F52-6 protein and application of coding gene of F52-6 protein and hydrolyzed xylan
  • F52-6 protein and application of coding gene of F52-6 protein and hydrolyzed xylan
  • F52-6 protein and application of coding gene of F52-6 protein and hydrolyzed xylan

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Experimental program
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Effect test

Embodiment 1

[0041] Cloning of embodiment 1, F52-6 gene

[0042] In the previous study, the metagenomic library of the dry fermented sludge system was constructed using the Fosmid vector. Using the Congo red-xylan plate, an Escherichia coli host clone containing xylanase activity was screened. The insert fragment contained in Fosmid is 38176bp, the ORF is predicted by softberry, and annotated by NCBI and Pfam database. Obtain the F52-6 gene, the nucleotide sequence of this gene is the 14th-1063 nucleotides from the 5' end of sequence 1 in the sequence listing, the protein encoded by it is named F52-6, and its amino acid sequence is the sequence in the sequence listing 2 Amino acids 1-351 from the N' terminal. After comparison, the F52-6 protein has the highest 56% similarity with the xylanase from Caldicoprobacter oshimai, and it is predicted to be a xylanase.

Embodiment 2

[0043] Embodiment 2, the application of F52-6 as xylanase

[0044] 1. Construction of recombinant vector

[0045] Using the artificially synthesized sequence 1 as a template, using F52-6F and F52-6R as primers, and using TAKARA's PrimeStar high-fidelity enzyme to perform PCR amplification, a 1075bp PCR product was obtained, and its nucleotide sequence was sequence 1.

[0046] F52-6F:5'-GGAATTC CATATG GAAGCTGGAGGTGTTCCCAT-3' contains Nde I restriction site

[0047] F52-6R:5'-CCC AAGCTT TTAGCGATCCTGTGCAGTGCT-3' contains Hind III restriction site

[0048] The above PCR amplification system is as follows:

[0049]

[0050]

[0051] The PCR program is as follows:

[0052]

[0053] The above PCR product was double digested with Nde I and Hind III, and the resulting digested product was ligated with the pET-28a vector (Novagen pET-28a DNA Cat. No. 69864-3) that had undergone the same digestion to obtain a recombinant vector.

[0054] After sequencing, the recombinant...

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Abstract

The invention discloses F52-6 protein and application of a coding gene of the F52-6 protein and hydrolyzed xylan. The protein disclosed by the invention is protein (1) or (2), wherein the protein (1) is formed by amino acid residues shown in a sequence 2 in a sequence table, and the protein (2) is formed through enabling a residue sequence of an amino acid sequence of the sequence 2 in the sequence table to be subjected to the substitution and / or deletion and / or addition of one or more amino acid residues and is protein which has the same functions and is derived from the protein (1). According to the F52-6 protein and the application of the coding gene of the F52-6 protein and the hydrolyzed xylan, proved by experiments, the optimal temperature for novel xylanase F52-6 is 75 DEG C under the conditions of taking xylan as a substrate and being in the pH value of 5.0, and activity of 82.3% can still be maintained after the novel xylanase F52-6 is incubated for 2 hours at the temperature of 70 DEG C; over 80% residual activity can be maintained in the pH value of 4.0-12.0 after the novel xylanase F52-6 is incubated for 24 hours in buffer solutions with different pH values at the temperature of 4 DEG C.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of F52-6 protein, its coding gene and hydrolyzed xylan. Background technique [0002] Xylan (Xylan) is an important component of hemicellulose in plant cell walls, accounting for about 35% of cell dry weight, and is an abundant renewable resource in nature, second only to cellulose. Xylanase (Xylananse) is a class of enzymes that can hydrolyze xylan into xylooligosaccharides and xylose. Many natural microorganisms contain xylanase. Because xylanase can be widely used in textile, paper, food and biofuel industries, it has attracted more and more research interests of scholars in recent years. In order to achieve high heat resistance and thermal stability of enzymes, many scholars screen new enzymes from thermophilic microorganisms, but they are rarely applicable to industrial conditions. In the biofuel industry, that is, the hydrolysis of biomass to produce bioethano...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N1/21C12N1/19C12N1/15C12P7/10C12P19/14
CPCC12N9/2437C12N9/2445C12N9/248C12P7/10C12Y302/01004C12Y302/01021Y02E50/10
Inventor 吴晓磊王蒙来国莉聂勇耿爽
Owner PEKING UNIV
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