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Cryophilic xylosidase/arabinofuranosidase and preparation method and application thereof

A technology of arabinosidase and xylosidase, which is applied in the field of cold-adapted xylosidase/arabinosidase bifunctional cellulose degrading enzyme and its preparation, and achieves the effects of improving utilization rate, less inhibitor and better degradation

Inactive Publication Date: 2011-05-11
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still few domestic related research reports. Only Xue Yemin and others cloned the enzyme Xar with this property from Thermoanaerobacter ethanolicus (Food and Fermentation Industry 29: 22-26, 2003)

Method used

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  • Cryophilic xylosidase/arabinofuranosidase and preparation method and application thereof
  • Cryophilic xylosidase/arabinofuranosidase and preparation method and application thereof
  • Cryophilic xylosidase/arabinofuranosidase and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Construction of yak rumen microbial metagenomic cosmid library and screening of xylosidase activity

[0047] The rumen samples of yaks in Qinghai, China were collected, filtered with 3 layers of gauze, and the filtrate was centrifuged to collect rumen microbial cells. For the extraction method of rumen microbial metagenome, please refer to An et al. ("Anaerobe" 2005, Volume 11, pages 207-215). For the construction method of the rumen-uncultured bacterial metagenomic library, refer to the product manual of the pWEB::TNC Cosmid Cloning Kit kit from Epicentre Company. The extracted metagenomic DNA was blunted with End-Repair Enzyme Mix, ligated with the dephosphorylated vector pWEB::TNC in the kit, and the ligated product was used MaxPlax TM After the Lambda Packaging Extracts were packaged, the host bacteria E.coli EPI100 was infected, and then spread on the LB-ampicillin plate, and cultured at 37°C for 12-16 hours to grow colonies, which were the metagenomic library of ...

Embodiment 2

[0048] Cloning and analysis of the sequence of the RuBGX1 gene of embodiment 2 rumen microorganism source

[0049] The screened cosmid-positive library clones were subcloned in pGEM11z, functionally screened, and sequenced. Specifically: the cosmid plasmid of the screened positive clone was partially digested with Sau3AI into a 2-5kb fragment, ligated into the pGEM11z vector that was digested with BamHI and dephosphorylated, and transformed into DH5α. The subclone library was screened for function, and the obtained subclone was sequenced with T7 and SP6 universal primers, and the sequence of the coding region of the β-1,4-xylosidase gene was analyzed by the analysis software DNAMAN, and the nucleotide sequence of the gene was determined such as SEQ ID NO 1 and named RuXyn1. RuXyn1 encodes 332 amino acids, its amino acid sequence is shown in SEQ ID NO.2, and its theoretical molecular weight is 42kDa. The protein domain of RuXyn1 was analyzed using SMART software (http: / / www.e...

Embodiment 3

[0051] Recombinant Expression of RuXyn1 Gene in Escherichia coli

[0052] The recombinant expression of RuXyn1 in Escherichia coli adopts the 6His fusion expression strategy, and the Escherichia coli expression vector pET-21a(+) (purchased from Novagen Company) is selected. Specifically: design and synthesize a set of oligonucleotide primers: forward primer 21axyn1FE: GGC GAATTC and reverse primer 21axyn1FX:CCG CTCGAG (The underlined parts in the sequence are the restriction endonucleases EcoRI and XhoI respectively, and the gene sequence is shown in italics), and the RuXyn1 gene is amplified by PCR, recovered by agarose electrophoresis and digested with EcoRI and XhoI respectively , connected with the vector pET-21a(+) digested with EcoRI and XhoI, the schematic diagram of the constructed RuXyn1 recombinant expression vector is as follows figure 1 shown. The ligation product was transformed into the Escherichia coli Top10 strain, and the obtained transformants were s...

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Abstract

The invention belongs to the field of biological engineering and provides a novel bifunctional enzyme RuXyn1 with low-temperature resistance and activities of beta-D-xylosidase and alpha-L-arabinofuranosidase. The RuXyn1 has an amino acid coding sequence shown as SEQ ID No 2 and has a nucleotide coding sequence shown as SEQ ID No 1. The RuXyn1 can be prepared by a genetic engineering method or an artificial synthesis method. The RuXyn1 has functions of the arabinofuranosidase and the xylosidase, can keep higher activity in a low-temperature environment, and can be applied to the application fields of cellulose bioconversion, chemical industry, textiles, foods, bioenergy, feed additives, pharmaceutical industry and the like.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to a bifunctional cellulose degrading enzyme of cold-adapted xylosidase / arabinosidase and its preparation method and application. The invention also relates to the recombinant expression plasmid and recombinant genetic engineering strain of the xylosidase / arabinosidase. Background technique [0002] Lignocellulose is an abundant renewable energy source in nature and consists of four main components: cellulose (~33-51%), hemicellulose (~19-34%), pectin (~2-20%) and woody Vegetarian (~20-30%). Degradation of lignocellulose produces fermentable hexose (glucose, 36-50%; mannose, 0.3-12%; galactose, 0.1-2.4%) and pentose (xylose, 3.4-23%) %; arabinose, 1.1 to 4.5%). The skeleton structure of hemicellulose is composed of β-1,4-xylan, and the degrading enzyme of xylan is mainly composed of endo-1,4-xylanase (endo-1,4-β-D-xylanase , EC3.2.1.8) and β-1,4-xylosidase (β-1,4-xylosidase, EC3.2.1....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N1/21C12N1/19C12N1/15C12N5/10A23K1/165C12P7/10C12P19/14C12R1/19C12R1/865C12R1/84C12R1/125A23K20/189
CPCY02E50/10
Inventor 吕红周峻岗袁汉英
Owner FUDAN UNIV
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