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Screening For Alcohol Dehydrogenase And Its Application On The Synthesis Of Montelukast

Posted on:2017-10-22Degree:MasterType:Thesis
Country:ChinaCandidate:D F YuFull Text:PDF
GTID:2381330488482333Subject:Biochemical Engineering
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Montelukast sodium is one of the main anti-asthmatic drugs.In synthesis of Montelukast process,enantiomerically pure methyl [E]-2-[3-(S)-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-hydroxyprop yl]benzoate is one of the key intermediates.In chemical synthesis of [E]-2-[3-(S)-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-hydroxyprop yl]benzoate,it needs low temperature,high pressure and expensive reagent.Biological catalytic synthesis has been gained lots of attentions for its advantages,such as,mild reaction condition,high reaction efficiency,cheap material,high conversion rate,high enantioselectivity ect.So it is necessary to research the biosynthesis process of [E]-2-[3-(S)-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-hydroxyprop yl]benzoate and improve its efficiency and productivity.In this work,a rapid reverse phase HPLC method for the detection of substrate(methyl 2-[3-[3-(2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-oxopropyl)benzoate)and product([E]-2-[3-(S)-[3-[2-(7-Chloro-2-quinolinyl)ethenyl]phenyl]-3-hydroxypropyl]benzoate)(following retention times: ketone 6.81 min and enantiomeric alcohol 5.80 min)and normal phase HPLC method(following retention times: ketone 11.24 min and enantiomeric alcohol 12.45 min)was established.The strain ZJB14004 with high conversion rate and high enantioselectivity was isolated from oxidoreductase database in the laboratory.Based on gene sequencing,blasting database and multiple sequence alignments analysis,we confirmed that the enzyme is ketoreductase and belongs to short-chain dehydrogenase.Secondly,the cultivation conditions of ZJB14004 were optimized.The conditions include pH,inoculum size,inducer type,final concentration of inducer,time to add inducer,induction time,induction temperature.The results showed that the optimal cultivation conditions were as follows: the inoculum size is 2%,seed age 6 h,2.5 h after inoculation add 2.5%(W/V)lactose and induce 11 h at 28 °C,respectively.Under the conditions above,the specific activity and biomass of ZJB14004 reached 670 U/g DCW and 4.13 g DCW/L which was 1.22-and 1.20-fold as compared to before optimization.The asymmetric reduction conditions of ZJB14004 were optimized.The conditions include type and ratio of organic,temperature,buffer pH,co-enzyme concentration,metal ion ect.The results showed that the optimal reaction conditions were as follows: toluene as the organic phase,the ratio of toluene is 5%,reaction temperature is 45 °C,pH 7.5 triethanolamine buffer(100 mM),0.1 g/L co-enzyme,respectively.During the feed-batch asymmetric reduction of 100 mL reaction system,the initial concentration of substrate was 65.5 mM,after 80 min and 160 min,65.5 mM and 87.3 mM substrate was added separately.The reaction lasting 5 h with the final conversion rate of 100%,product 218.3 mM,e.e.>99.9%,space time yield 43.7 mM/h,1.4-fold higher than one time feeding space time yield.Finally,the extraction process of product was explored,the yield of product was 45.7%,and the product purity was >99%.
Keywords/Search Tags:Intermediates of montelukast, alcohol dehydrogenase, asymmetric reduction, product separation
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