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
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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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