caa a22 4500 605500029 CHVBK 20210128100553.0 cr unu---uuuuu 210128e20150501xx s 000 0 eng 10.1007/s00253-015-6527-z doi (NATIONALLICENCE)springer-10.1007/s00253-015-6527-z An efficient high-throughput screening assay for rapid directed evolution of halohydrin dehalogenase for preparation of β-substituted alcohols [Elektronische Daten] [Nan-Wei Wan, Zhi-Qiang Liu, Feng Xue, Kai Huang, Ling-Jiao Tang, Yu-Guo Zheng] Halohydrin dehalogenases (HHDHs) are an important class of enzymes for preparing opticallyactive haloalcohols, epoxides, and β-substituted alcohols. However, natural HHDHs rarely meet the requirements of industrial applications. Here, a novel high-throughput screening (HTS) methodology for directed evolution of HHDH was developed based on the colorimetric determination of azide. In this method, azide was involved in the HHDH-catalyzed ring-opening process and the decrease of azide was used to quantitatively evaluate HHDH activity. The HTS methodology was simple and sensitive (ε460 = 1.2173 × 104Lmol−1cm−1) and could be performed in a microplate format using whole cells. To verify the efficiency of the HTS methodology, it was adopted to engineer a HHDH (HHDH-PL) from Parvibaculum lavamentivorans DS-1, which was applied in the process for ethyl (R)-4-cyano-3-hydroxybutanoate (HN) by the conversion of ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE)). A random mutant library containing 2500 colonies was screened using the HTS methodology, and three beneficial mutants F176M, A187R, and A187S were obtained. By combining the beneficial mutated residues, the variant F176M/A187R was identified with 2.8-fold higher catalytic efficiency for preparation of HN. The high-throughput colorimetric assay would be very useful for directed evolution of HHDH for preparing β-substituted alcohols. Springer-Verlag Berlin Heidelberg, 2015 Halohydrin dehalogenase nationallicence High-throughput screening nationallicence Colorimetric assay nationallicence Ethyl ( S )-4-chloro-3-hydroxybutanoate nationallicence Ethyl ( R )-4-cyano-3-hydroxybutanoate nationallicence Wan Nan-Wei Institute of Bioengineering, Zhejiang University of Technology, 310014, Hangzhou, People's Republic of China aut Liu Zhi-Qiang Institute of Bioengineering, Zhejiang University of Technology, 310014, Hangzhou, People's Republic of China aut Xue Feng Institute of Bioengineering, Zhejiang University of Technology, 310014, Hangzhou, People's Republic of China aut Huang Kai Institute of Bioengineering, Zhejiang University of Technology, 310014, Hangzhou, People's Republic of China aut Tang Ling-Jiao Institute of Bioengineering, Zhejiang University of Technology, 310014, Hangzhou, People's Republic of China aut Zheng Yu-Guo Institute of Bioengineering, Zhejiang University of Technology, 310014, Hangzhou, People's Republic of China aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/9(2015-05-01), 4019-4029 0175-7598 99:9<4019 2015 99 253 https://doi.org/10.1007/s00253-015-6527-z 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/s00253-015-6527-z text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Wan Nan-Wei Institute of Bioengineering, Zhejiang University of Technology, 310014, Hangzhou, People's Republic of China aut NATIONALLICENCE 700 1- Liu Zhi-Qiang Institute of Bioengineering, Zhejiang University of Technology, 310014, Hangzhou, People's Republic of China aut NATIONALLICENCE 700 1- Xue Feng Institute of Bioengineering, Zhejiang University of Technology, 310014, Hangzhou, People's Republic of China aut NATIONALLICENCE 700 1- Huang Kai Institute of Bioengineering, Zhejiang University of Technology, 310014, Hangzhou, People's Republic of China aut NATIONALLICENCE 700 1- Tang Ling-Jiao Institute of Bioengineering, Zhejiang University of Technology, 310014, Hangzhou, People's Republic of China aut NATIONALLICENCE 700 1- Zheng Yu-Guo Institute of Bioengineering, Zhejiang University of Technology, 310014, Hangzhou, People's Republic of China aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/9(2015-05-01), 4019-4029 0175-7598 99:9<4019 2015 99 253