A kind of acidophilic α-galactosidase agalb with galactomannan degrading ability and its gene and application

A technology of galactomannan and galactosidase, which is applied in the field of genetic engineering and can solve problems such as inability to hydrolyze various substrates

Active Publication Date: 2011-12-28
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the food and feed industry, the added α-galactosidase is required to have high hydrolysis ability to various α-galactoside oligosaccharides, and the substrate of the previously reported α-galactosidase Specificity cannot hydrolyze a variety of substrates, each has its limitations in application

Method used

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  • A kind of acidophilic α-galactosidase agalb with galactomannan degrading ability and its gene and application
  • A kind of acidophilic α-galactosidase agalb with galactomannan degrading ability and its gene and application
  • A kind of acidophilic α-galactosidase agalb with galactomannan degrading ability and its gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Cloning of the gene AgalB encoding the acidophilic fungus Bispora sp.MEY-1α-galactosidase

[0046] Extraction of acidophilic fungus Bispora sp.MEY-1 genomic DNA:

[0047] Filter the mycelium cultured in liquid for 3 days with sterile filter paper, put it into a mortar, add 2mL of extract, grind for 5min, then put the grinding solution in a 50mL centrifuge tube, lyse in a water bath at 65°C for 20min, and mix every 10min. Homogenize once and centrifuge at 10,000 rpm for 5 min at 4°C. The supernatant was extracted in phenol / chloroform to remove impurity proteins, 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 vacuum, dissolved by adding an appropriate amount of TE, and stored at -20°C for later use.

[0048] Through the construction of the cDNA library...

Embodiment 2

[0052] RT-PCR analysis of embodiment 2α-galactosidase gene

[0053] Extract the total RNA of Bispora sp.MEY-1, use reverse transcriptase to obtain a strand of cDNA, and then design appropriate primers (AgalB F1: 5′-ATGGCGATACTTTTTGCTTGCTTTGCGACGCTTG-3′, AgalB R: 5′-TCATACCGAACATTGATAGTAGAAGGCGCATGTGC-3′) The single-stranded cDNA was amplified to obtain the cDNA sequence of α-galactosidase, and the amplified product was recovered and sent to Shanghai Sangong for sequencing.

[0054] By comparing the genome sequence and cDNA sequence of α-galactosidase, it is found that the gene has 3 introns, the cDNA is 1281bp long, encodes 426 amino acids and a stop codon, and the N-terminal 17 amino acids are its predicted signal peptide Sequence, the measured nucleotide sequence of the mature protein part of the gene AgalB is homologously compared with the α-galactosidase gene sequence on GeneBank, the highest amino acid sequence identity is 35%, and the nucleotide sequence identity is 36% ...

Embodiment 3

[0055] Example 3 Preparation of recombinant α-galactosidase.

[0056] The expression vector pPIC9 was subjected to double digestion (SnaBI+NotI), and the gene AgalB encoding α-galactosidase was double-digested (SnaBI+NotI) at the same time, and the gene fragment encoding mature α-galactosidase was cut out and expressed The vector pPIC9 was ligated to obtain the recombinant plasmid pPIC-AgalB containing the Bispora sp.MEY-1α-galactosidase gene AgalB and transformed into Pichia pastoris GS115 to obtain the recombinant Pichia pastoris strain GS115 / AgalB.

[0057] The GS115 strain containing the recombinant plasmid was inoculated in 400mL of BMGY culture medium, shaken at 250rpm at 30°C for 48h, and then collected by centrifugation. Then resuspend in 200mL BMMY medium, shake culture at 20°C and 250rpm. After 72 hours of induction, the supernatant was collected by centrifugation. The activity of α-galactosidase was determined. The expression level of recombinant α-galactosidase ...

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Abstract

The invention relates to the field of genetic engineering, in particular, the invention relates to an acidophilic alpha-galactosidase AgalB with galactomannan degrading ability and its gene and application. According to the α-galactosidase AgalB of the present invention, it has the amino acid sequence shown in SEQ ID NO.1 or 3, and the above-mentioned α-galactosidase has the nucleotide sequence shown in SEQ ID NO.4 or 6 . The present invention provides a new α-galactosidase gene, the α-galactosidase encoded by it has acidophilicity, wide action pH range, better heat resistance, stronger protease resistance and better The ability to hydrolyze various substrates can be used in industries such as feed, food, and medicine. According to the technical scheme of the invention, the production of acidophilic α-galactosidase with galactomannan degrading ability can be realized by means of genetic engineering.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to an acidophilic alpha-galactosidase AgalB with galactomannan degrading ability and its gene and application. Background technique [0002] α-galactosidase or melibisidase (α-galactosidase, α-D-galactoside galactohydrolase, EC 3.2.1.22) is an exoglycosidase that converts non-reducing The terminal α-(1,6) bonded galactosyl group is catalyzed by hydrolysis. Such glycosides include galactooligosaccharides (such as melibiose, raffinose and stachyose), branched polysaccharides (such as galactomannan) and galactolipids (Margolles-Clark E, et al. Eur J Biochem, 1996, 240(1): 104-11.). [0003] α-Galactosidase has been widely used in the feed industry as a new type of feed enzyme preparation. Soybean meal is the most used protein raw material in feed. The content of raffinose and stachyose in soybean meal is about 7%. They cannot be degraded by endogenous enzyme...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/40C12N15/56C12N15/63C12N1/19C12R1/645C12R1/84C12R1/865C12R1/78
Inventor 姚斌罗会颖王慧王亚茹柏映国黄火清石鹏君袁铁铮杨培龙孟昆
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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