Development of Novel Lyases and Their Application to the Synthesis of Optically Active Compounds

首先，从节杆菌属细菌中筛选出具有催化顺丁烯二酸和衣康酸水合反应的新型水解酶的微生物，以生产食品添加剂D-苹果酸和柠檬酸等。纯化至均一，并对其进行了鉴定。研究了以马来酸为原料生产光学纯D-苹果酸的培养条件和反应条件。在最佳反应条件下，20h内D-苹果酸的产量为87g/L，产率为72%。对(S)-(+)-柠檬酸盐产柠檬酸的活性进行了研究。脱氮假单胞菌产生(S)-(+)-柠檬酸对映体过量99.9%。在最佳反应条件下，30h每升可合成(S)-(+)-柠檬酸27g，产率为69mol%。其次，对催化富马酸衍生物合成L-天冬氨酸衍生物的新型3-甲基天冬氨酸解氨酶进行了筛选。我们首次发现3-甲基天冬氨酸解氨酶(MAL)产生菌在兼性厌氧肠杆菌家族中分布较广。对弗劳迪柠檬酸杆菌(Citrobacter Freundii)、柠檬柠檬酸杆菌(C.amalonaticus)和摩根摩根氏杆菌(Mganella Mganii)的结晶物质进行了表征。利用这些菌株的无细胞提取物，由相应的富马酸衍生物合成了光学纯的(2S，3S)-3-甲基天冬氨酸、(2S，3S)-3-乙基天冬氨酸和(2R，3S)-3-氯天冬氨酸。

First, we screened for microorganisms having new hydro-lyases catalyzing hydration of maleic acid and itaconic acid, respectively, to produce D-malic acid and citramalic acid which are food additives and starting materials in synthetic organic chemistry, etc. The bacterial maleate hydratase from Arthrobacter sp. was purified to homogeneity and characterized. The culture and reaction conditions for the production of optically pure D-malate from maleate were studied. Under an optimum condition, 87 grams/liter of D-malate was produced in 20 h. The yield was 72 mole %. (S)-(+)-Citramalate producing activity from itaconate was also studied. A.denitrificans produced (S)-(+)-Citramalate in a 99.9 % enantiomeric excess. Under an optimum condition, 27 g of (S)-(+)-citramalate per liter was produced in 30 h. The yield was 69 mol%.Second, a screening was carried out for new 3-methylaspartate ammonia-lyases which catalyze the synthesis of L-aspartic acid derivatives from fumaric acid derivatives. We discovered for the first time that 3-methylaspartate ammonia-lyase (MAL) producers are relatively widely distributed in the family of facultatively anaerobic Enterobacteriaceae. Crystalline MALs from Citrobacter freundii, C.amalonaticus, and Morganella morganii, were characterized. Using cell-free extracts of the isolates, optically pure (2S, 3S)-3-methylaspartic, (2S, 3S)-3-ethylaspartic, and (2R, 3S)-3-chloroaspartic acids were synthesized from corresponding fumaric acid derivatives.