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芳香族L-氨基酸是合成许多药物、农药、精细化学品和食品添加剂的重要手性砌块(Chiral buildingblocks)。利用酶催化具有高活性和高立体选择性的特点合成手性砌块是目前不对称合成领域重要的研究方向。通过对不同来源转氨酶的进化分析,选择分别源自原核生物大肠杆菌Escherichia coli和真核生物酿酒酵母Saccharomyces cerevisia中的两种具有代表性Ⅰ型芳香族转氨酶TyrB和Aro8,比较研究了两种转氨酶通过平衡逆转不对称氨化催化合成芳香族L-氨基酸的反应过程和催化效率。重组转氨酶TyrB和Aro8都能有效地合成天然芳香族氨基酸苯丙氨酸和酪氨酸以及非天然氨基酸苯甘氨酸。手性HPLC分析表明,合成的氨基酸都是L-构型的,e.e值等于100%。L-丙氨酸是适宜的氨基供体,转氨酶TyrB和Aro8都不能利用D-型氨基酸作为氨基供体。反应体系中氨基供体L-丙氨酸和氨基受体芳香族α-酮酸的最适摩尔比为4∶1。底物芳香族α-酮酸分子结构中芳香环上的取代基以及脂肪酸碳链部分的长度都对酶催化的转氨效率有显著的影响。在制备规模试验中,TyrB催化不对称转氨反应合成L-苯甘氨酸、L-苯丙氨酸和L-酪氨酸的比生产速率为0.28 g/(g.h)、0.31 g/(g.h)和0.60 g/(g.h),Aro8催化上述反应的比生产速率分别为0.61 g/(g.h)、0.48 g/(g.h)和0.59 g/(g.h)。研究结果对利用转氨酶通过平衡逆转不对称催化合成芳香族L-氨基酸的工业化应用具有指导意义。
Aromatic L-amino acids are important chiral building blocks for the synthesis of many drugs, pesticides, fine chemicals and food additives. The use of enzyme-catalyzed synthesis of chiral building blocks with high activity and high stereoselectivity is an important research field in the field of asymmetric synthesis. Based on the evolutionary analysis of aminotransferases from different sources, TyrB and Aro8, one of the representative type I aromatic transaminases, were isolated from Escherichia coli and Saccharomyces cerevisia respectively. The effects of two aminotransferases Balanced reversal of asymmetric amination catalyzed synthesis of aromatic L-amino acid reaction and catalytic efficiency. Both recombinant transaminases TyrB and Aro8 efficiently synthesize the natural aromatic amino acids phenylalanine and tyrosine, as well as the unnatural amino acid phenylglycine. Chiral HPLC analysis showed that all the synthesized amino acids were in the L-configuration with an e.e value equal to 100%. L-alanine is a suitable amino donor, and both transaminases TyrB and Aro8 can not utilize D-amino acids as amino donors. The optimum molar ratio of the amino donor L-alanine to the amino acceptor aromatic α-keto acid in the reaction system is 4: 1. Substituents on the aromatic rings in the aromatic a-keto acid structure of the substrate and the length of the carbon chain of the fatty acid all have a significant effect on the enzyme-catalyzed transamination efficiency. In the preparation scale test, TyrB catalyzed the asymmetric transamination reaction to synthesize L-phenylglycine. The specific production rates of L-phenylalanine and L-tyrosine were 0.28 g / (gh), 0.31 g / (gh) and The specific productivity of 0.68 g / (gh) and 0.68 g / (gh) for Aro8 catalyzed the above reaction were 0.61 g / (gh), 0.48 g / (gh) and 0.59 g / (gh), respectively. The results of this study are instructive for the industrial application of the transaminase to reverse the asymmetric synthesis of aromatic L-amino acids.