A biological preparation method of (s)-5-chloro-2,3-dihydro-2-hydroxyl-1-oxo-1h-indene-2-carboxylic acid methyl ester
A technology of methyl carboxylate and biological preparation, applied in the direction of fermentation, etc., can solve the problems of economical and environmental friendliness, limited actual sources, high price, etc., and achieve high ee value, high conversion rate, and environmental friendliness Effect
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Embodiment 1
[0022] In a 2mL centrifuge tube, add 975μL 100mM pH 8.0 phosphate buffer solution in sequence, add substrate 5-chloro-2,3-dihydro-1-oxo-1H-indene-2-carboxylic acid methyl ester 2mg, methanol 25 μL, after stirring evenly, add 2 mg of different monooxygenases (from Suzhou Hanzyme Biotechnology Co., Ltd.), 2 mg of NADP, 3.2 mg of glucose, 2 mg of glucose dehydrogenase, shake the reaction at 30 ° C, and detect the reaction results by HPLC in 24 hours As in Table 1.
[0023] Table 1
[0024] EW1531 EW1535 EW1536 EW1538 Conversion rates 33％ 48.3％ 47.3％ 43.9％ product ee 96.1％ 96.7％ 92.5％ 100％
[0025] It can be seen from Table 1 that under the same other reaction conditions, the monooxygenase with brand EW1535 has the highest substrate conversion rate, and the monooxygenase with brand EW1538 has the highest e value of the product.
Embodiment 2
[0027] In a 5mL centrifuge tube, add 1950μL of 100mM pH 8.0 phosphate buffer solution successively, add substrate 5-chloro-2,3-dihydro-1-oxo-1H-indene-2-carboxylate 4mg, methanol 50 μL, after stirring evenly, add 1 mg of monooxygenase EW1538 (from Suzhou Hanzyme Biotechnology Co., Ltd.), 0.5 mg of NADP, 6 mg of glucose, 1 mg of glucose dehydrogenase, and shake the reaction at different temperatures. The results of HPLC detection and reaction are shown in Table 2 .
[0028] Table 2
[0029] temperature / ℃ 20 30 40 1h 27.9％ 24.5％ 7.0％ 3h 57.5％ 67.6％ 7.5％ 24h 67.6％ 71.4％ 7.6％
[0030] It can be seen from Table 2 that under the same other reaction conditions, the conversion rate of the substrate is the highest when the reaction is performed at a temperature of 30°C.
Embodiment 3
[0032] In a 5mL centrifuge tube, add 1950 μL of 100 mM buffer solution with different pH in sequence, add 4 mg of substrate 5-chloro-2,3-dihydro-1-oxo-1H-indene-2-carboxylate, 50 μL of methanol, After stirring evenly, 1 mg of monooxygenase EW1538 (from Suzhou Hanzyme Biotechnology Co., Ltd.), 0.5 mg of NADP, 6 mg of glucose, and 1 mg of glucose dehydrogenase were added, and the reaction was shaken at 30°C. The results of the reaction detected by HPLC are shown in Table 3.
[0033] table 3
[0034] pH 5.5 6.0 7.0 8.0 1h 14.9％ 20.7％ 27.0％ 24.5％ 3h 23.9％ 39.8％ 72.8％ 67.6％ 24h 19.4％ 33.6％ 80.1％ 71.4％
[0035] As can be seen from Table 3, under the same conditions as other reaction conditions, the above reaction is carried out in an aqueous buffer solution with a pH of 7.0, and the conversion rate of the substrate is the highest.
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