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High specific activity xylosidase Xyl52B8 and gene and application thereof

A technology of live xylosidase and xylosidase, applied in high specific activity xylosidase Xyl52B8 and its gene and application field, can solve the problems of poor thermal stability and unsatisfactory, and achieve good thermal stability and high activity effect

Active Publication Date: 2013-03-20
WUHAN SUNHY BIOLOGICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Industrial production requires enzymes to undergo short-term high-temperature treatment. At present, the optimum temperature of most xylosidases is around 50 degrees Celsius, but the poor thermal stability cannot meet the industrial requirements of feed pelleting, brewing and processing.

Method used

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  • High specific activity xylosidase Xyl52B8 and gene and application thereof
  • High specific activity xylosidase Xyl52B8 and gene and application thereof
  • High specific activity xylosidase Xyl52B8 and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 Cloning of Alicyclobacillus sp.B8 Xylosidase Encoding Gene xyl52

[0079] Extract Genomic DNA of Alicyclobacillus sp.B8: Extract the bacterial cells cultured in liquid for 1 day with a bacterial extraction kit, add appropriate amount of TE to dissolve, and store at -20°C for later use.

[0080] The degenerate primers P1 and P2 were designed and synthesized according to the conserved (GNEMDG and DEWNVW) sequences of the xylosidase genes of the 52nd family

[0081] P1: 5'-GGNAAYGARATGGAYGG-3';

[0082] P2: 5'-GGAEITTYD SLDVSLGQ-3'.

[0083] Alicyclobacillus sp.B8 total DNA was used as template for PCR amplification. The PCR reaction parameters were: denaturation at 94°C for 5 min; then denaturation at 94°C for 30 sec, annealing at 50°C for 30 sec, extension at 72°C for 1 min, and after 30 cycles, incubation at 72°C for 10 min. A fragment of about 2 kb was obtained, which was recovered and connected with the pEASY-T3 vector and sent to Sanbo Biotechnology Co.,...

Embodiment 2

[0088] The preparation of embodiment 2 recombinant xylosidase

[0089] The expression vector pPET-30a was subjected to double enzyme digestion (Ecoli I+Not I), and the gene xyl52B8 encoding xylosidase was double enzyme digested (Ecoli I+Not I) at the same time, and the gene fragment encoding mature xylosidase was cut out and expressed The vector pPET-30a was ligated to obtain the recombinant plasmid pPET-xyl52B8 containing the xylosidase gene xyl52B8 and transformed into Escherichia coli BL21(DE3) to obtain the recombinant Escherichia coli BL21 / xyl52B8.

[0090] The BL21 strain containing the recombinant plasmid was inoculated in 300mL LB culture medium, shaken at 220rpm at 37°C for 3h, and then induced with a final concentration of 0.6mM IPTG at 30°C for 4h. The expression level of recombinant xylosidase was 69.86U / mL. The results of SDS-PAGE showed that the recombinant xylosidase was expressed in Escherichia coli. The specific activity of recombinant xylan is 200.7U / mg.

Embodiment 3

[0091] The activity analysis of embodiment 3 recombinant xylosidases

[0092] Determination of xylosidase activity: measure the amount of p-nitrophenol produced by enzymatic hydrolysis of substrate pNPX at 405 nm. Reaction steps: Mix 250 μL of 2mM pNPX substrate with 150 μL of buffer, add 100 μL of appropriately diluted enzyme solution, react at 50°C for 10 min, add 1.5 mL of 1M Na2CO3 to terminate the reaction, and measure the OD value at 405 nm.

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Abstract

The present invention relates to the field of genetic engineering, and specifically relates to specific activity xylosidase Xyl52B8 and gene and application thereof. The amino acid sequence of the xylosidase Xyl52B8 is shown as SEQ ID No. 1. Firstly, the technical problem to be solved by the present invention is to overcome the deficiencies in the prior art, and the invention provides novel xylosidase which has excellent nature, and is suitable for application in the feed and food industries. The optimum pH of the xylosidase of the present invention is 6.0. The xylosidase has relatively high enzyme activity at pH 5.0-7.0, and good stability of pH. The high specific activity of the xylosidase enables the application thereof in the industrial production in need of high-temperature environment.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a high specific activity xylosidase Xyl52B8 and its gene and application. Background technique [0002] Xylan is the most abundant class of hemicelluloses and is widely found in hardwoods (15-30%), softwoods (7-10%) and herbaceous plants (less than 30%). The basic sugar unit of xylan is d-xylopyranose. Its main chain is formed by connecting xylose with β-1,4 glycosidic bonds, and the side chains are modified by various substituents. At the same time, these side chains are The chain substituents are cross-linked to each other by chemical bonds, forming complex structures (Collins et al. FEMS Microbiology Reviews. 2005, 29:3-23.). The degradation of xylan requires the synergistic action of main chain hydrolase and branch chain hydrolase, main chain enzymes include β-1,4-xylanase and β-xylosidase, side chain hydrolysis requires α-l-arabinofuran Glycosidase...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N1/19C12R1/84
Inventor 詹志春陶纯长方萍
Owner WUHAN SUNHY BIOLOGICAL
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