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High-temperature resistance arabinfuranosidease Abf51B8, as well as gene and application thereof

A furanosidase and high temperature-resistant technology, applied in the field of genetic engineering, can solve the problems of poor thermal stability and inability to meet the requirements

Active Publication Date: 2012-10-10
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 arabinofuranosidases is around 50°C, but their poor thermal stability cannot meet the high-temperature production requirements of industries such as feed granulation and brewing processing.

Method used

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  • High-temperature resistance arabinfuranosidease Abf51B8, as well as gene and application thereof
  • High-temperature resistance arabinfuranosidease Abf51B8, as well as gene and application thereof
  • High-temperature resistance arabinfuranosidease Abf51B8, as well as gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Cloning of Alicyclobacillus sp.B8 Arabinofuranosidase Encoding Gene abf51B8

[0042] Extract the genomic DNA of Alicyclobacillus sp.B8:

[0043] Centrifuge the bacteria cultured in the liquid for 3 days, put them into a mortar, add 2mL extract, grind for 5min, then put the grinding solution in a 50mL centrifuge tube, lyse in a water bath at 65°C for 20min, mix well every 10min, and Centrifuge at 10,000 rpm for 5 min at 4°C. The supernatant was extracted in phenol / chloroform to remove impurities, 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 vacuo, dissolved by adding an appropriate amount of TE, and stored at -20°C for later use.

[0044] The degenerate primers P1, P2 were designed and synthesized according to the conserved (GNEMDG and DEWNVW) seque...

Embodiment 2

[0052] The preparation of embodiment 2 recombinant arabinofuranosidase

[0053] The expression vector pPET-30a is subjected to double digestion (Nde I+Not I), and the gene abf51B8 encoding arabinofuranosidase is double-digested (Nde I+Not I) at the same time, and the gene fragment encoding mature arabinofuranosidase is cut out Ligated with the expression vector pPET-30a, the recombinant plasmid pPET-abf51B8 containing the arabinofuranosidase gene abf51B8 was obtained and transformed into Escherichia coli BL21(DE3) to obtain the recombinant Pichia pastoris strain BL21 / abf51B8.

[0054] The BL21 strain containing the recombinant plasmid was inoculated in 300mL LB culture medium, shaken at 227rpm at 37°C for 2h, and then induced with a final concentration of 0.6mM IPTG at 30°C for 4h. The expression level of recombinant arabinofuranosidase was 5.6U / mL. The results of SDS-PAGE showed that the recombinant arabinofuranosidase was expressed in Escherichia coli. The specific activit...

Embodiment 3

[0055] Example 3 Activity analysis of recombinant arabinofuranosidase

[0056] Determination of the activity of arabinofuranosidase: the amount of the product p-nitrophenol generated by the enzymatic hydrolysis of the substrate pNPAf was measured at 405 nm. Reaction steps: Mix 250μL 2mM pNPAf substrate with 150μL buffer, add 100μL appropriately diluted enzyme solution, react at 40°C for 10min, add 1.5mL 1M Na 2 CO 3 The reaction was terminated, and the OD value was measured at 405 nm.

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Abstract

The invention relates to the field of gene engineering, and in particular relates to high-temperature resistance arabinfuranosidease Abf51B8, as well as a gene and an application thereof. The invention provides the arabinfuranosidease Abf51B8 coming from Alicyclobacillussp.B8 with an amino acid sequence shown as SEQ ID NO.1, and a coding gene abf51B8 for coding the arabinfuranosidease. The arabinfuranosidease has the characteristics of optimal pH of 6.0, optimal temperature of 60 DEG C and good thermal stability at 70 DEG C, and can be widely applied to feeds, foods, energy industries and the like serving as a novel enzyme preparation.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a high-temperature-resistant arabinofuranosidase Abf51B8 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 synergy of main chain hydrolase and branch chain hydrolase, the main chain enzyme includes β-1,4-xylanase and β-xylosidase, and the side chain hydrolysis requires α-L-arabinofura...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/56C12N15/63C12N1/15C12N1/19C12N1/21
Inventor 詹志春顾爱玲
Owner WUHAN SUNHY BIOLOGICAL
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