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Acidophilic alpha-galactosidase AgalB with galactomannan degradation capability and gene and application thereof

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

Active Publication Date: 2010-09-01
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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  • Acidophilic alpha-galactosidase AgalB with galactomannan degradation capability and gene and application thereof
  • Acidophilic alpha-galactosidase AgalB with galactomannan degradation capability and gene and application thereof
  • Acidophilic alpha-galactosidase AgalB with galactomannan degradation capability and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0046] Extract genomic DNA from the acidophilic fungus Bispora sp.MEY-1:

[0047] Filter the mycelium cultured for 3 days with sterile filter paper and put it in a mortar, add 2 mL of extract, grind for 5 min, then place the grind in a 50 mL centrifuge tube, lyse in a water bath at 65°C for 20 min, and mix every 10 min. Homogenize once and centrifuge at 10,000 rpm at 4°C for 5 min. The supernatant was extracted in phenol / chloroform to remove impurities, and then the supernatant was added to an equal volume of isopropanol. After standing at room temperature for 5 min, centrifuged at 10,000 rpm at 4°C for 10 min. The supernatant was discarded, the precipitate was washed twice with 70% ethanol, dried in vacuum, dissolved in an appropriate amount of TE, and placed at -20°C for use.

[0048] Through the construction of cDNA library, EST sequencing and Blast comparison, the partial nu...

Embodiment 2

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

[0053] Extract the total RNA of Bispora sp.MEY-1, use reverse transcriptase to obtain a strand of cDNA, and then design the 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 Shenggong for sequencing.

[0054] By comparing the genome sequence and cDNA sequence of α-galactosidase, it was found that the gene has 3 introns. The cDNA is 1281bp long, encoding 426 amino acids and a stop codon, and 17 amino acids at the N-terminal are its predicted signal peptide Sequence, the determined partial nucleotide sequence of the mature protein of the gene AgalB is 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 double digested (SnaBI+NotI), and the gene encoding α-galactosidase AgalB was double digested (SnaBI+NotI), and the gene fragment encoding mature α-galactosidase was cut out and expressed The vector pPIC9 was connected 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] Take the GS115 strain containing the recombinant plasmid, inoculate it in 400 mL of BMGY culture solution, culture with shaking at 250 rpm at 30°C for 48 hours, and collect the bacteria by centrifugation. Then it was resuspended in 200 mL BMMY medium and cultured with shaking at 250 rpm at 20°C. After 72 hours of induction, the supernatant was collected by centrifugation. Determine the activity of α-galactosidase. The expression level of recombinant α...

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Abstract

The invention relates to the field of genetic engineering, in particular to acidophilic alpha-galactosidase AgalB with galactomannan degradation capability and a gene and application thereof. The alpha-galactosidase AgalB has an amino acid sequence expressed by SEQ ID No. 1 or 3, and the coded alpha-galactosidase has an amino acid sequence expressed by SEQ ID No. 4 or 6. The invention provides a new alpha-galactosidase gene, wherein the alpha-galactosidase coded by the alpha-galactosidase gene has acidophilic property, wide acting pH range, good heat resistance, strong proteinase resistance and good capability of hydrolyzing various substrates and can be applied to the industries of feed, food, medicament and the like. The technical scheme of the invention can realize production of the acidophilic alpha-galactosidase with galactomannan degradation capability by using genetic engineering measures.

Description

Technical field [0001] The present invention relates to the field of genetic engineering, in particular, the present invention relates to an acidophilic alpha-galactosidase AgalB with galactomannan degradation ability and its gene and application. Background technique [0002] α-galactosidase or melibiase (α-galactosidase, α-D-galactoside galactohydrolase, EC 3.2.1.22) is an exo-type glycosidase, which can remove non-reducing properties from various galactosidase The galactosyl group linked to the terminal α-(1,6) bond catalyzes the 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] As a new type of feed enzyme preparation, α-galactosidase has been widely used in the feed industry. Soybean meal is the most used protein material in feed. The content of raffinose and stachyose in soybean...

Claims

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Application Information

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Patent Type & Authority Applications(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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