Imabayashi Yuki, Suzuki Shun'ichi, Kawasaki Hisashi, Nakamatsu Tsuyoshi
a AminoScience Laboratories , Ajinomoto Co., Ltd. , Kawasaki , Japan.
b Department of Environmental Materials Science , Tokyo Denki University , Adachi-ku , Japan.
Biosci Biotechnol Biochem. 2016;80(1):104-13. doi: 10.1080/09168451.2015.1072458. Epub 2015 Aug 19.
For the production of enantiopure β-amino acids, enantioselective resolution of N-acyl β-amino acids using acylases, especially those recognizing N-acetyl-β-amino acids, is one of the most attractive methods. Burkholderia sp. AJ110349 had been reported to exhibit either (R)- or (S)-enantiomer selective N-acetyl-β-Phe amidohydrolyzing activity, and in this study, both (R)- and (S)-enantioselective N-acetyl-β-Phe acylases were purified to be electrophoretically pure and determined the sequences, respectively. They were quite different in terms of enantioselectivities and in their amino acids sequences and molecular weights. Although both the purified acylases were confirmed to catalyze N-acetyl hydrolyzing activities, neither of them show sequence similarities to the N-acetyl-α-amino acid acylases reported thus far. Both (R)- and (S)-enantioselective N-acetyl-β-Phe acylase were expressed in Escherichia coli. Using these recombinant strains, enantiomerically pure (R)-β-Phe (>99% ee) and (S)-β-Phe (>99% ee) were obtained from the racemic substrate.
对于对映体纯β-氨基酸的生产，使用酰基化酶对N-酰基β-氨基酸进行对映选择性拆分，尤其是那些识别N-乙酰基-β-氨基酸的酰基化酶，是最具吸引力的方法之一。据报道，伯克霍尔德氏菌AJ110349表现出（R）-或（S）-对映体选择性N-乙酰基-β-苯丙氨酸酰胺水解活性，在本研究中，（R）-和（S）-对映选择性N-乙酰基-β-苯丙氨酸酰基化酶均被纯化至电泳纯，并分别测定了序列。它们在对映选择性、氨基酸序列和分子量方面有很大差异。尽管纯化的两种酰基化酶均被证实具有催化N-乙酰基水解的活性，但它们与迄今为止报道的N-乙酰基-α-氨基酸酰基化酶均无序列相似性。（R）-和（S）-对映选择性N-乙酰基-β-苯丙氨酸酰基化酶均在大肠杆菌中表达。使用这些重组菌株，从外消旋底物中获得了对映体纯的（R）-β-苯丙氨酸（ee>99%）和（S）-β-苯丙氨酸（ee>99%）。
