An organic solvent-resistant galactosidase high-yielding bacterium and the gene and application of the galactosidase
A technology of galactosidase and resistance to organic solvents, applied in the field of biopharmaceuticals, can solve the problems of low product molar conversion rate, high price, low substrate concentration, etc., and achieve the effect of good application potential
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Embodiment 1
[0039] This example illustrates the screening procedure for organic solvent-resistant strains of galactosylation-modified nucleoside drugs.
[0040] The following method is used for primary screening: with X-Gal (5-bromo-4-chloro-3- indole -β-D-galactoside) as the substrate, inoculate the strains in the organic solvent-resistant bacterial library in our laboratory to the screening plate, and directly observe the color change of the plate, if it turns blue, it means that the strain has β-galactoside Glycosidase hydrolytic activity. The specific screening medium formula is: lactose 10 g / L, yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, MgSO 4 0.5 g / L, K 2 HPO 4 2.5g / L, X-Gal 0.1g / L, pH 7.0, agar 20g / L. The culture temperature is 37 ℃, and the culture time is 12-36 h. This method can screen a large number of organic solvent-resistant β-galactosidase-producing bacteria.
[0041] The strains obtained from the primary screening were re-screened, and the specific method ...
Embodiment 2
[0043] This example illustrates the biological properties and identification of the organic solvent-resistant galactosidase-producing bacterium YZ09.
[0044] Biological properties of the strain YZ09: the strain is a Gram-positive bacterium without spores. The bacterial body is a bacillus under the microscope, facultatively anaerobic, and the optimum growth temperature is 30-40 ℃. Its physiological and biochemical characteristics are manifested in: catalase reaction results are negative, gelatin reaction results are negative, no exercise, glucose is oxidized to produce acid, and lactose, sucrose, sorbitol, xylose, fructose, maltose, mannitol, etc. can be utilized.
[0045] After 16S rDNA sequence analysis, strain YZ09 was identified as Lactobacillus casei ( Lactobacillus casei ), named Lactobacillus casei ( Lactobacillus casei ) YZ09.
[0046] Lactobacillus casei ( Lactobacillus casei ) YZ09, has been preserved. Classified as Lactobacillus casei ( Lactobacillus casei ...
Embodiment 3
[0048] This example illustrates Lactobacillus casei ( Lactobacillus casei ) YZ09 fermentation enzyme production method.
[0049] Plate medium formula: peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, glucose 20 g / L, K 2 HPO 4 2 g / L, sodium zirconate 5 g / L, citric acid 2 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.2 g / L, agar 20 g / L, pH6.5, soluble in water.
[0050] Seed medium formula: peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, glucose 20 g / L, K 2 HPO 4 2 g / L, sodium zirconate 5 g / L, citric acid 2 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.2 g / L, pH6.5, soluble in water.
[0051] Seed solution preparation: The volume of seed solution medium in a 250 mL Erlenmeyer flask was 40 mL. The strain YZ09 cultured on the plate for 24 h was scraped and inserted into the seed medium, the rotation speed of the shaker was 180 rpm, the culture temperature was 37 °C, and the culture time was 10-12 h to obtain the seed liquid.
[0052] Enzyme ...
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