caa a22 4500 605503850 CHVBK 20210128100613.0 cr unu---uuuuu 210128e20150201xx s 000 0 eng 10.1007/s00253-014-5992-0 doi (NATIONALLICENCE)springer-10.1007/s00253-014-5992-0 Double substituted variant of Bacillus amyloliquefaciens esterase with enhanced enantioselectivity and high activity towards 1-(3′,4′-methylenedioxyphenyl)ethyl acetate [Elektronische Daten] [Jia-Yan Liu, Han-Ping Bian, Yun Tang, Yun-Peng Bai, Jian-He Xu] Bacillus amyloliquefaciens esterase (BAE) was applied to produce (R)-1-(3′,4′-methylenedioxyphenyl)ethanol, a chiral drug intermediate. In this study, we improved the enantioselectivity of BAE by protein engineering instead of process engineering as used in our previous work. Saturation mutagenesis was carried out on eight positions of BAE based on structure modeling and substrate docking. A double substituted variant V10 (K358D/A396C) showed an excellent enantioselectivity without decreasing the activity. The functions of these two mutations (K358D and A396C) were investigated, revealing a synergic effect on the BAE enantioselectivity. Using the variant V10, enantiopure (R)-1-(3′,4′-methylenedioxyphenyl)ethanol could be readily prepared in >97% ee, affording a high space-time yield (123gL−1 day−1) and a high ratio of substrate/catalyst (40gg−1) in 1-L reaction. Springer-Verlag Berlin Heidelberg, 2014 Bacillus amyloliquefaciens nationallicence Esterase nationallicence Enantioselectivity nationallicence Iterative saturation mutagenesis nationallicence 1-(3′,4′-Methylenedioxyphenyl)ethanol nationallicence Liu Jia-Yan State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, 200237, Shanghai, China aut Bian Han-Ping Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Meilong Road, 200237, Shanghai, China aut Tang Yun Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Meilong Road, 200237, Shanghai, China aut Bai Yun-Peng State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, 200237, Shanghai, China aut Xu Jian-He State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, 200237, Shanghai, China aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/4(2015-02-01), 1701-1708 0175-7598 99:4<1701 2015 99 253 https://doi.org/10.1007/s00253-014-5992-0 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-014-5992-0 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Liu Jia-Yan State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, 200237, Shanghai, China aut NATIONALLICENCE 700 1- Bian Han-Ping Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Meilong Road, 200237, Shanghai, China aut NATIONALLICENCE 700 1- Tang Yun Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Meilong Road, 200237, Shanghai, China aut NATIONALLICENCE 700 1- Bai Yun-Peng State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, 200237, Shanghai, China aut NATIONALLICENCE 700 1- Xu Jian-He State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, 200237, Shanghai, China aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/4(2015-02-01), 1701-1708 0175-7598 99:4<1701 2015 99 253