An efficient high-throughput screening assay for rapid directed evolution of halohydrin dehalogenase for preparation of β-substituted alcohols
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| 001 | 605500029 | ||
| 003 | CHVBK | ||
| 005 | 20210128100553.0 | ||
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| 008 | 210128e20150501xx s 000 0 eng | ||
| 024 | 7 | 0 | |a 10.1007/s00253-015-6527-z |2 doi |
| 035 | |a (NATIONALLICENCE)springer-10.1007/s00253-015-6527-z | ||
| 245 | 0 | 3 | |a An efficient high-throughput screening assay for rapid directed evolution of halohydrin dehalogenase for preparation of β-substituted alcohols |h [Elektronische Daten] |c [Nan-Wei Wan, Zhi-Qiang Liu, Feng Xue, Kai Huang, Ling-Jiao Tang, Yu-Guo Zheng] |
| 520 | 3 | |a 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. | |
| 540 | |a Springer-Verlag Berlin Heidelberg, 2015 | ||
| 690 | 7 | |a Halohydrin dehalogenase |2 nationallicence | |
| 690 | 7 | |a High-throughput screening |2 nationallicence | |
| 690 | 7 | |a Colorimetric assay |2 nationallicence | |
| 690 | 7 | |a Ethyl ( S )-4-chloro-3-hydroxybutanoate |2 nationallicence | |
| 690 | 7 | |a Ethyl ( R )-4-cyano-3-hydroxybutanoate |2 nationallicence | |
| 700 | 1 | |a Wan |D Nan-Wei |u Institute of Bioengineering, Zhejiang University of Technology, 310014, Hangzhou, People's Republic of China |4 aut | |
| 700 | 1 | |a Liu |D Zhi-Qiang |u Institute of Bioengineering, Zhejiang University of Technology, 310014, Hangzhou, People's Republic of China |4 aut | |
| 700 | 1 | |a Xue |D Feng |u Institute of Bioengineering, Zhejiang University of Technology, 310014, Hangzhou, People's Republic of China |4 aut | |
| 700 | 1 | |a Huang |D Kai |u Institute of Bioengineering, Zhejiang University of Technology, 310014, Hangzhou, People's Republic of China |4 aut | |
| 700 | 1 | |a Tang |D Ling-Jiao |u Institute of Bioengineering, Zhejiang University of Technology, 310014, Hangzhou, People's Republic of China |4 aut | |
| 700 | 1 | |a Zheng |D Yu-Guo |u Institute of Bioengineering, Zhejiang University of Technology, 310014, Hangzhou, People's Republic of China |4 aut | |
| 773 | 0 | |t Applied Microbiology and Biotechnology |d Springer Berlin Heidelberg |g 99/9(2015-05-01), 4019-4029 |x 0175-7598 |q 99:9<4019 |1 2015 |2 99 |o 253 | |
| 856 | 4 | 0 | |u https://doi.org/10.1007/s00253-015-6527-z |q text/html |z Onlinezugriff via DOI |
| 898 | |a BK010053 |b XK010053 |c XK010000 | ||
| 900 | 7 | |a Metadata rights reserved |b Springer special CC-BY-NC licence |2 nationallicence | |
| 908 | |D 1 |a research-article |2 jats | ||
| 949 | |B NATIONALLICENCE |F NATIONALLICENCE |b NL-springer | ||
| 950 | |B NATIONALLICENCE |P 856 |E 40 |u https://doi.org/10.1007/s00253-015-6527-z |q text/html |z Onlinezugriff via DOI | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Wan |D Nan-Wei |u Institute of Bioengineering, Zhejiang University of Technology, 310014, Hangzhou, People's Republic of China |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Liu |D Zhi-Qiang |u Institute of Bioengineering, Zhejiang University of Technology, 310014, Hangzhou, People's Republic of China |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Xue |D Feng |u Institute of Bioengineering, Zhejiang University of Technology, 310014, Hangzhou, People's Republic of China |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Huang |D Kai |u Institute of Bioengineering, Zhejiang University of Technology, 310014, Hangzhou, People's Republic of China |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Tang |D Ling-Jiao |u Institute of Bioengineering, Zhejiang University of Technology, 310014, Hangzhou, People's Republic of China |4 aut | ||
| 950 | |B NATIONALLICENCE |P 700 |E 1- |a Zheng |D Yu-Guo |u Institute of Bioengineering, Zhejiang University of Technology, 310014, Hangzhou, People's Republic of China |4 aut | ||
| 950 | |B NATIONALLICENCE |P 773 |E 0- |t Applied Microbiology and Biotechnology |d Springer Berlin Heidelberg |g 99/9(2015-05-01), 4019-4029 |x 0175-7598 |q 99:9<4019 |1 2015 |2 99 |o 253 | ||