Engineered ketoreductase polypeptide and method for preparing montelukast midbody by means of same
A compound, ketopropyl technology, applied in the field of engineered ketoreductase polypeptides and preparation of montelukast intermediates, can solve the problems of low substrate concentration, long reaction time, poor atom economy, etc., and achieve the substrate concentration High, overcoming the effect of poor reaction performance and short reaction time
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Embodiment 1
[0020] Embodiment 1 (preparation of ketoreductase):
[0021] A ketoreductase catalyst was prepared using conventional methods: Sequence in the table 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43 gene fragments were provided by Suzhou Jinweizhi Biology Synthesized by Science and Technology Co., Ltd., connected with pET30a (Novagen) plasmid enzyme digestion product, transformed into competent E.coli BL21 (DE3) strain, screened to obtain positive clones, inoculated into liquid LB medium containing resistance, at 37 Cultivate at ℃ until OD600 to 0.8, add inducer IPTG, continue to cultivate for 16 hours, centrifuge to collect the precipitate, add phosphate buffer to suspend, ultrasonically break in ice-water bath for 10 minutes, centrifuge to take the supernatant, and freeze to obtain ketoreductase enzyme powder.
Embodiment 2
[0022] Embodiment 2 (screening of ketoreductase)
[0023] By sequence 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43 respectively in Escherichia coli Ketoreductase 2 mg obtained by expression, glucose dehydrogenase (purchased from Suzhou Hanzyme Biotechnology Co., Ltd., brand EW002) 2 mg, NADP 1 mg, substrate 2-(3-(3-((E)-2-(7 -Chloroquinolinyl)vinyl)phenyl)-3-ketopropyl)methyl benzoate 50mg and glucose 50mg were added to a 5mL reactor containing 2mL 0.05M triethanolamine buffer solution, stirred at 30°C at 1000rpm, after 1h Sampling by HPLC showed that the conversion rate and ee of sequence 43 were both >99%, so sequence 43 was used as the object of further research.
Embodiment 3
[0024] Embodiment 3 (enzyme catalyzed reaction):
[0025] Add 100 mg of ketoreductase (obtained by expression of sequence 43 in Escherichia coli), 20 mg of glucose dehydrogenase (purchased from Suzhou Hanzyme Biotechnology Co., Ltd., brand EW002) and 5 mg of NADP triethanolamine buffer into a 50 mL three-necked reaction flask in sequence. solution, 5g substrate 2-(3-(3-((E)-2-(7-chloroquinolinyl)vinyl)phenyl)-3-ketopropyl)methyl benzoate, 3.5g glucose, 20mL 0.05M pH 7.0 triethanolamine buffer, 5mL toluene, temperature adjusted to 30°C, stirring at 900r / min, 15%Na 2 CO 3 The pH of the solution was maintained at 7.0, reacted for 12 hours, and the conversion rate was 99% as detected by HPLC, added 30ml of methanol, refluxed at 60°C for 2h, filtered and added deionized water dropwise to the filtrate until no solid precipitated, filtered to take the filter cake and added at 60°C After the methanol was just dissolved, deionized water was added dropwise until no solid precipitated,...
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