CAJ | 학술논문

生物催化具有反应条件温和、绿色环保、高立体选择性等优点，已成为制备光学活性度洛西汀的重要方式和途径，但仍存在有效的生物催化剂数量有限和反应效率低等问题，基因组数据库的发展为新型立体选择性生物催化剂的开发提供了有效的途径和平台。本研究通过基因组信息挖掘，从 Candida parapsilosis CCTCCM 203011中发现了8个新型的醛酮还原酶，分析研究不同的醛酮还原酶对N,N-二甲基-3-酮-3-(2-噻吩)-1-丙胺(DKTP)的催化还原能力，发现 CPAR4能够高立体选择性地催化还原 DKTP 生成度洛西汀中间体(S)-N,N-二甲基-3-羟基-3-(2-噻吩)-1-丙胺(DHTP)。通过粗酶体系反应参数的优化，CPAR4催化还原底物浓度在3g/L 时，产率达到94.5%，光学纯度大于99.9%ee；当底物浓度为5g/L 时，产率仍达到70%以上。该重组菌粗酶液能够高立体选择性地生物还原DKTP生成度洛西汀中间体(S)-DHTP，具有一定的应用潜力。
생물최화구유반응조건온화、록색배보、고입체선택성등우점，이성위제비광학활성도락서정적중요방식화도경，단잉존재유효적생물최화제수량유한화반응효솔저등문제，기인조수거고적발전위신형입체선택성생물최화제적개발제공료유효적도경화평태。본연구통과기인조신식알굴，종 Candida parapsilosis CCTCCM 203011중발현료8개신형적철동환원매，분석연구불동적철동환원매대N,N-이갑기-3-동-3-(2-새분)-1-병알(DKTP)적최화환원능력，발현 CPAR4능구고입체선택성지최화환원 DKTP 생성도락서정중간체(S)-N,N-이갑기-3-간기-3-(2-새분)-1-병알(DHTP)。통과조매체계반응삼수적우화，CPAR4최화환원저물농도재3g/L 시，산솔체도94.5%，광학순도대우99.9%ee；당저물농도위5g/L 시，산솔잉체도70%이상。해중조균조매액능구고입체선택성지생물환원DKTP생성도락서정중간체(S)-DHTP，구유일정적응용잠력。
Biocatalytic synthesis of chiral Duloxetine and its intermediate is one of the greatest technologies due to its high chemo-,regio- and stereo-selectivity;while the amount of available biocatalyst is yet limited and the conversion efficiency needs to be improved. In recent years,the availability of microbial genome sequences allows scientists to discover novel enzymes with potential applications. Based on the genome sequence of Candida parapsilosis,eight aldo-keto reductases were discovered and evaluated for the synthesis of N,N-dimethyl-3-keto-3-(2-thienyl)-1- propanamine (DKTP). Among them , CPAR4 showed higher catalytic ability for asymmetric reduction of N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine (DKTP) to (S)-N,N-dimethyl-3-hydroxy-3-(2-thienyl)-1-propanamine (DHTP),a key intermediate of chiral Duloxetine. Then the biomediated reaction conditions using the cell-free system involving the expressed CPAR4 were optimized and (S)-DHTP (>99.9%ee) was produced with the yield of 94.5%at the initial substrate concentration of 3g/L,and even when the substrate concentration was increased to 5g/L,the enantiopure product can also be obtained with the yield over 70%. Therefore,the newly identified enzyme and its cell-free system would be promising for highly stereo specific bioreduction of prochiral pharmaceutical intermediates.