Tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate prepared by biological catalysis and strain
A strain, the technology of ZJB-09225, is applied in the field of biocatalytic preparation of tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate and strains, and can solve the problems of insufficient diastereomeric induction and the like
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Embodiment 1
[0036] Example 1: Screening microbial strains with catalytic (R)-6-cyano-5-hydroxyl-3-oxoylhexanoic acid tert-butyl ester reduction activity
[0037] Soil samples collected from all over the country were dispersed with normal saline in a certain proportion and then inoculated into enrichment medium containing 0.40g / L (R)-6-cyano-5-hydroxy-3-oxohexanoic acid tert-butyl ester (Preparation: Boil 50.0g glucose, 100.0g soybean sprouts for 30 minutes, add 0.40g (R)-6-cyano-5-hydroxy-3-oxoylhexanoic acid tert-butyl ester, add water to make up to 1.0L, do not adjust the pH ), under the conditions of 30°C and 150rpm, culture on a shaking table until the culture medium becomes turbid, and then transfer to 0.80g / L (R)-6-cyano-5 - in the rich medium of tert-butyl hydroxy-3-oxohexanoate, continue to culture at 30°C and 150rpm. The concentration of tert-butyl (R)-6-cyano-5-hydroxy-3-oxohexanoate in the enrichment medium was successively increased until it reached 2.0 g / L.
[0038] The las...
Embodiment 2
[0041] Example 2: Effect of the initial pH value of the fermentation medium on the enzyme activity of P.caribbic ZJB-09225 carbonyl reductase volume
[0042] For the P.caribbic ZJB-09225 bacteria stored on the slant of the test tube, pick an inoculation loop and inoculate it into the sterile fermentation medium. The composition of the fermentation medium is as follows: malt extract powder 30.0g / L, glucose 20.0g / L, CuSO 4 3.0mg / L, KH 2 PO 4 1.36g / L, K 2 HPO 4 ·3H 2 O2.28g / L, NaCl1.0g / L, the solvent is water, the temperature is 28°C, the initial pH value of the fermentation medium is 5.0, 6.0, 7.0, 8.0, 9.0, and cultured at 150rpm for 2 days, and the fermentation broth is collected.
[0043] Centrifuge the fermentation broth at 12000rpm for 8 minutes, discard the supernatant, collect the cells, wash 3 times with normal saline, weigh 1g of the wet cells, and then use 10.0mL, pH7.0 phosphate buffer (50mM) to wash the cells Disperse in a transformation bottle, add 0.14g (R)-6...
Embodiment 3
[0044] Example 3: The influence of culture temperature on the enzyme activity of P.caribbic ZJB-09225 carbonyl reductase volume
[0045] For the P.caribbic ZJB-09225 bacteria stored on the slant of the test tube, pick an inoculation loop and inoculate it into the sterile fermentation medium. The composition of the fermentation medium is as follows: malt extract powder 30.0g / L, glucose 20.0 g / L, CuSO 4 3mg / L, KH 2 PO 4 1.36g / L, K 2 HPO 4 ·3H 2 O2.28g / L, NaCl1.0g / L, solvent is water, pH7.0, cultured at 23°C, 28°C, 30°C, 32°C, 35°C, 37°C, 150rpm for 2 days, collected fermentation broth.
[0046] Centrifuge the fermentation broth at 12000rpm for 8 minutes, discard the supernatant, collect the cells, wash 3 times with normal saline, weigh 1g of the wet cells, and then disperse the washed cells with 10.0mL, pH7.0 phosphate buffer (50mM) In the transformation bottle, add 0.14g (R)-6-cyano-5-hydroxy-3-oxoylhexanoic acid tert-butyl ester and 0.20g glucose, place in a water bath ...
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