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Neutral alpha-galactosidase Aga-S27 having high degradability of alpha-galactoside oligosaccharide, and genes and application thereof

A technology of galactosidase and aga-s27, which is applied in the field of genetic engineering and can solve problems such as inability to hydrolyze various substrates

Inactive Publication Date: 2010-09-22
JIANGSU YINONG BIOLOGY CO LTD
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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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  • Neutral alpha-galactosidase Aga-S27 having high degradability of alpha-galactoside oligosaccharide, and genes and application thereof
  • Neutral alpha-galactosidase Aga-S27 having high degradability of alpha-galactoside oligosaccharide, and genes and application thereof
  • Neutral alpha-galactosidase Aga-S27 having high degradability of alpha-galactoside oligosaccharide, and genes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1 Isolation of Streptomyces sp.S27 and its enzyme-producing properties

[0094] Strain Streptomyces sp.S27 was isolated from the soil of Huoyan Mountain in Xinjiang. After growing on Gaoshi No. 1 medium for 2 days, the bacteria aggregated into white balls. The primers designed according to the conserved sequence of bacterial 16S rDNA (27F: 5′-AGAGTTTGATCMTGGCTCAG-3′ and 1492R: 5′-TACGGHTACCTTACGACTT-3′) were used for PCR amplification of the 16S rDNA of the strain, and the sequencing results were compared with the nucleosides in the Genbank database. According to acid sequence comparison, the 16S rDNA nucleotide sequence of S27 has the highest similarity of 100% with Streptomyces radiopugnans strain HBUM174051 (Genbank accession No. FJ486333). Combined with morphological observation, it can be proved that the strain is Streptomyces sp.S27. Soybean meal was added as an inducer and carbon source to the enzyme-producing medium (0.1% K 2 HPO 4 , 0.3% NaNO 3 , 0....

Embodiment 2

[0095] Cloning of Example 2 Streptomyces α-Galactosidase Encoding Gene aga-S27

[0096] Extract the genomic DNA of Streptomyces sp.S27: Centrifuge the bacterial solution cultured in Gao’s No. 1 medium at 30°C for 2 days at 10,000 rpm for 10 minutes, weigh 50 mg of bacterial sludge and wash with 500 μL of sterile water, and centrifuge to get the precipitated bacteria. body. Resuspend the precipitated bacteria in 500 μL of lysozyme mixture, incubate at 37°C for 1 hour, add 100 μL of enzyme solution and continue to incubate at 45°C for 30 minutes until the bacteria solution is transparent, add 10% SDS to a final concentration of 2%, and stir for about 5 minutes Until the viscosity of the bacterial liquid drops significantly, centrifuge at 15,000 rpm for 10 minutes to remove debris. The supernatant was extracted with equal volumes of phenol, phenol:chloroform, and chloroform in sequence. Take the upper layer solution and add 0.6-1 times the volume of isopropanol to precipitate a...

Embodiment 3

[0099] Activity analysis of embodiment 3α-galactosidase

[0100] Enzyme activity was determined by the pNPG method. Dissolve pNPG in 0.1mol / L McIlvaine buffer to make the final concentration 2mmol / L. Mix 20 μL of enzyme solution, 230 μL of McIlvaine buffer and 250 μL of 2 mmol / L pNPG, and shake well. After incubating at 37°C for 5 min, add 1.5 mL of 1 mol / L Na to the reaction solution 2 CO 3 solution to terminate the reaction. The OD value was measured at 405nm, and the enzyme activity was expressed by the production of p-nitrophenol (pNP). After adding enzyme solution and buffer to the control tube, add Na first 2 CO 3 The solution was then added with pNPG solution.

[0101] Enzyme activity (U / mL) unit definition: The amount of enzyme needed to decompose pNPG to release 1 μmol pNP per minute at 37°C is defined as one enzyme activity unit.

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Abstract

The invention relates to the field of genetic engineering and particularly provides neutral alpha-galactosidase Aga-S27 having high degradability of alpha-galactoside oligosaccharide, and genes and application thereof. The neutral alpha-galactosidase Aga-S27 having high degradability of alpha-galactoside oligosaccharide can be obtained from a bacterial strain, i.e., novel streptomyces sp.S27 (CGMCC No.3146) screened by the invention, and the genes thereof and recombinant vector containing the genes can be further obtained, wherein the amino acid sequences of alpha-galactosidase Aga-S27 are shown in SEQ ID No.1. The alpha-galactosidase Aga-S27 has the advantages of appropriate operating temperature and pH value, higher protease resistance and higher ability to hydrolyze various substrates. Therefore, the alpha-galactosidase Aga-S27 is applicable as feed or food additives in the industries of feed and food.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a neutral alpha-galactosidase Aga-S27 with high alpha-galactoside oligosaccharide degrading ability and its gene, a recombinant vector containing the gene and application thereof. Background technique [0002] α-galactosidase (α-galactosidase, EC3.2.1.22) is melibiase, which belongs to the class of exoglycosidases and can specifically catalyze the hydrolysis of α-galactosidic bonds at the non-reducing ends of sugar chains. Not only can it hydrolyze oligosaccharides containing α-galactosidic bonds, but it can also catalyze polysaccharides containing such bonds. Raffinose, stachyose, and verbascose are oligosaccharides widely present in legumes. Under the action of α-galactosidase, these oligosaccharides can be decomposed into D-galactose and other corresponding monosaccharides. sugars and oligosaccharides. [0003] Soybean meal is a very good plant-based...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/40C12N15/56C12N15/63C12N1/21C12N1/19A23K1/165A23L1/305C12R1/645C12R1/225C12R1/07C12R1/19C12R1/465
Inventor 项有炜王娟娟张慧君曹雅男
Owner JIANGSU YINONG BIOLOGY CO LTD
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