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High temperature-resistant acid arabinfuranosidease AbfaHLB and gene and application thereof

A furanosidase and high temperature-resistant technology, applied in the field of genetic engineering, can solve the problems of insufficiency and poor thermal stability, and achieve the effects of good thermal stability and good stability

Active Publication Date: 2016-07-20
北京科为博生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

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  • High temperature-resistant acid arabinfuranosidease AbfaHLB and gene and application thereof
  • High temperature-resistant acid arabinfuranosidease AbfaHLB and gene and application thereof
  • High temperature-resistant acid arabinfuranosidease AbfaHLB and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Cloning of the Arabinofuranosidase Gene abfaHLB in Example 1 Bacillus badiusHLB

[0047] Extraction of Bacillusbadius HLB genomic DNA:

[0048] (1) Take 0.5-2mL culture liquid, centrifuge at 10000rpm for 30s, absorb the supernatant as much as possible, and collect the bacteria;

[0049] (2) Add 200 μL buffer RB to the EP tube to resuspend, centrifuge at 10,000 rpm for 30 s, and discard the supernatant;

[0050] (3) For Gram-positive bacteria: add 120 μL lysozyme, mix by inverting, and bathe in 37°C water bath for 30-60 minutes;

[0051] (4) Centrifuge at 12000rpm for 2min, discard the supernatant and resuspend the cells in 180μL buffer RB by shaking or pipetting;

[0052] (5) Add 20 μL of RNaseA (25 mg / mL) solution, vortex and mix, and place at room temperature for 5-10 min;

[0053] (6) Add 800 μL of binding solution CB, then add 100 μL of isopropanol, and immediately vortex to mix thoroughly, at this time flocculent precipitation may appear;

[0054] (7) Add the m...

Embodiment 2

[0065] The preparation of embodiment 2 arabinofuranosidase AbfaHLB

[0066] Design and synthesize expression primers according to the obtained gene sequence of arabinofuranosidase AbfaHLB:

[0067] P3: 5'-GATGAATTCATGTCTATGGATGTAGATCCACGTTTA-3';

[0068] P4: 5'-GCTGCGGCCGCTACATTTACACGTAAACGAATCCAAGA-3'.

[0069] Using the total DNA of Bacillus badius HLB as a template, PCR amplification was performed again to obtain the gene of arabinofuranosidase AbfaHLB with a recombination restriction site. The expression vector pPIC9 is subjected to double digestion (EcoRI+NotI), and the gene abfaHLB encoding arabinofuranosidase is double-digested (EcoRI+NotI) at the same time, and the gene fragment encoding mature arabinofuranosidase is digested and connected to the expression vector pPIC9 , obtain the recombinant plasmid pPIC-abfaHLB containing the Bacillusbadius HLB arabinofuranosidase gene abfaHLB and transform Pichia pastoris GS115 by electroporation to obtain the recombinant Pichia...

Embodiment 3

[0071] Example 3 Activity analysis of recombinant arabinofuranosidase AbfaHLB

[0072] 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 250 μL 2mM pNPAf substrate with 150 μL buffer, add 100 μL appropriately diluted enzyme solution, react at 40°C for 10 min, add 1.5 mL 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, in particular to a high temperature-resistant acid arabinfuranosidease AbfaHLB and a gene and an application thereof. The arabinfuranosidease AbfaHLB comes from bacillus badius HLB, an amino acid sequence is shown in SEQ ID No.1, and an encoding gene abfa HLB for encoding the arabinfuranosidease AbfaHLB is provided. The arabinfuranosidease has the advantages that the following properties are realized, the suitable pH (potential of hydrogen) value is 4.5, the suitable temperature is 50 DEG C, and the thermal stability at the temperature of 80 DEG C is good; the arabinfuranosidease is used as a new enzyme preparation, and can be widely applied to feeds, foods, energy source industry and the like.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a high-temperature-resistant acid arabinofuranosidase AbfaHLB and its gene, a recombinant vector containing the gene and application thereof. Background technique [0002] 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 to form complex structures. Xylan is the most abundant polysaccharide in hemicellulose, widely present in hardwood (15-30%), softwood (7-10%) and herbaceous plants (less than 30%). The degradation of xylan requires the synergy of the main chain hydrolase and branch chain hydrolase, the main chain hydrolase includes β-1,4-xylanase and β-xylosidase, and the side chain hydrolase needs α-l- Arabinofu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/81C12N1/19C12R1/84
CPCC12N9/2402C12Y302/01055
Inventor 吴培均李富伟罗建杰李兆勇
Owner 北京科为博生物科技有限公司
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