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Anti-protease acidic alpha-galactosidase Aga-F75 and gene and application thereof

A technology of galactosidase and aga-f75, which is applied in the field of genetic engineering and can solve problems such as inability to hydrolyze various substrates

Active Publication Date: 2011-09-21
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

There are many kinds of oligosaccharides and polysaccharides containing α-galactosidic bonds, but the substrate specificity of the previously reported α-galactosidase cannot hydrolyze a variety of substrates, and each has its limitations in application

Method used

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  • Anti-protease acidic alpha-galactosidase Aga-F75 and gene and application thereof
  • Anti-protease acidic alpha-galactosidase Aga-F75 and gene and application thereof
  • Anti-protease acidic alpha-galactosidase Aga-F75 and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0152] Example 1 Isolation of Gibberela.sp.F75 and its enzyme-producing properties

[0153] After the strain Gibberela.sp.F75 grew on PDA medium for 3-5 days, the hyphae were white and the roots were red. Primers designed according to the conserved sequence of 18S rDNA of filamentous fungi were used to amplify the 18S rDNA of the strain by PCR, and the sequencing results were compared with the nucleotide sequences in the Genbank database. accession No.AB237662) has the highest similarity of 99%. Combined with morphological observation, it can be proved that the strain is Rhizopus, named Gibberella.sp.F75. Soybean meal was added as an inducer and carbon source to the enzyme-producing medium (4% K 2 HPO 4 , 0.28% (NH 4 ) 2 SO 4 , 0.12% CaCl 2 , 0.12% Urea, 0.12% MgSO 4 , 0.02% Mannanose, 0.02% Yeast extract, 3% soybean meal as carbon source), 30°C, 250rpm shaking culture for 5 days, the bacterial liquid was centrifuged, and the supernatant was taken to measure the enzyme...

Embodiment 2

[0154] Example 2 Cloning of gibberella α-galactosidase coding gene aga-F75

[0155] Extraction of Gibberela (Gibberela.sp.F75) genomic DNA: centrifuge the Gibberela liquid cultured at 30°C for 7 days at 6000rpm for 10min. Take 100mg of mycelia and add 500μL of sterile water to wash, centrifuge to get the precipitate. The precipitate was resuspended in 500 μL extract mixture, incubated at 37°C for 60 min, and centrifuged at 10,000 rpm for 10 min to remove the precipitate. The supernatant was extracted sequentially with equal volumes of phenol, phenol:chloroform, and chloroform. Take the upper layer solution and add 0.6-1 times the volume of isopropanol to precipitate at room temperature for 10 minutes. Centrifuge at 12000rpm for 15min. The precipitate was washed with 70% ethanol, centrifuged slightly, dried and dissolved in 30 μL sterile water for later use.

[0156] According to the published conserved sequence of α-galactosidase, degenerate primers P1, 5′-GAYGAYGGNTGGTTYG...

Embodiment 3

[0158] Example 3 Activity analysis of α-galactosidase.

[0159] 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 2mM pNPG, and shake well. After incubating at 37°C for 5 min, 1.5 mL of 1M Na was added 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.

[0160] 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 genetic engineering field, especially to a novel gibberella Gibberela.sp. F75 with a preservation number CGMCC No.2499, and an acidic alpha-galactosidase Aga-F75 of antiprotease from said strain, further a gene thereof and a recombinant vector containing said gene. The amino acid sequence of the alpha-galactosidase Aga-F75 is shown as SEQ ID NO.1. The enzyme has a proper action pH value, a stronger protease resistance and a better hydrolysis ability for various substrates and is used for the feed and food industries as a feed or food additive agent.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a protease-resistant acid α-galactosidase Aga-F75 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 protein feed raw material, generally used in the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12R1/225C12N15/63C12N9/40C12R1/19A23K1/165C12N1/14C12N1/19C12N1/21C12R1/07C12N15/56A23L1/30C12R1/645A23K20/189
Inventor 姚斌杨培龙曹雅男孟昆王亚茹罗会颖柏映国石鹏君袁铁铮史秀云
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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