大肠杆菌氨基转移酶的非天然氨基酸底物特异性研究

Study on the substrate specificity of non-natural amino acids ofEscherichia coli aminotransferase

氨基转移酶催化L-氨基酸和α-酮酸之间的氨基转移.TLC(薄层层析)和甲臜颜色反应检测大肠杆菌4种氨基转移酶(TyrAT、IlvAT、AspAT和AvtAT)催化6种非天然氨基酸的转氨活性,发现AvtAT和AspAT无非天然氨基酸转氨活性.TyrAT能催化DL-高苯丙氨酸、L-正亮氨酸和L-正缬氨酸的转氨反应,而IlvAT则催化DL-高苯丙氨酸、L-正缬氨酸、L-正亮氨酸、L-叔亮氨酸和L-新戊基甘氨酸的氨基转移.使用易错PCR突变和DL-高苯丙氨酸/L-正亮氨酸作氮源定向进化筛选,获得10个不同位点突变的TyrAT突变酶.酶活分析发现,这些突变酶虽酶活不同但尚未扩展TyrAT的非天然氨基酸底物特异性.10个突变酶中,每个酶蛋白210位都出现Cys替换Phe.与野生型酶比较,TyrAT的F210C突变明显改变酶的催化最适温度并且提高酶在37℃的催化效率.

Aminotransferases catalyze aminotransferation between L-amino acid andα-ketoacid.Transamination activities of E.coli aminotransferases(TyrAT,IlvAT,AspAT and AvtAT)towards 6 non-natural amino acids were tested by thin layer chromatography(TLC)and formazan color reaction.The results showed:1)AspAT and AvtAT did not catalyze aminotransferation of 6 non-natural amino acids;2)TyrAT catalyzed aminotransferation of DL-homophenylalanine,L-norleucine and L-norvaline;3)IlvAT catalyzed aminotransferation of DL-homophenylalanine,L-norleucine,L-norvaline,L-tertleucine and L-neropentylglycine.The tyrB gene was mutated by error-prone PCR and directed evolution in which DL-homophenylalanine or L-norleucine was used as nitrogen source,and 10 mutants were finally selected.Assay of enzyme activity showed that all mutant enzymes displayed different activities but did not broaden the substrate specificity of TyrAT.In 10 mutated enzymes tested,F210 of each enzyme was replaced by Cys.F210C mutation reduced the optimum temperature of enzyme catalysis and raised the catalytic efficiency of TyrAT at 37℃as compared to wild-type enzyme.