当前位置： SCI文献检索 > INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES期刊下所有文献 > Heterologous overexpression of a novel halohydrin dehalogenase from Pseudomonas pohangensis and modification of its enantioselectivity by semi-rational protein engineering.

Heterologous overexpression of a novel halohydrin dehalogenase from Pseudomonas pohangensis and modification of its enantioselectivity by semi-rational protein engineering.

Abstract：

:A gene encoding a halohydrin dehalogenase from Pseudomonas pohangensis (PpHHDH) was identified, synthesized and expressed in Escherichia coli. Subsequently, we used protein engineering to enhance the enzyme's enantioselectivity. We created two enantiocomplementary HHDH mutants, N160L and Q159L, which exhibited higher S- and R-selectivity toward PGE, respectively. The exchange of Leu at 159 for Gln led to a 2.3-fold increase in enantioselectivity (E-value of 22.2) compared to the wild-type. In addition, the N160L mutant displayed an inverted enantioselectivity (from ER = 9.8 to ES = 21.6) toward PGE. The wild-type PpHHDH and its variants were purified and characterized. They all displayed maximum activity at pH 7.5. The optimum temperature of mutant Q159L and N160L was increased from 35 °C to 40 °C. The wild-type PpHHDH and N160L mutant had good pH stability at pH 5.0-7.5, and Q159L showed an even wider range of pH tolerance, from pH 4.5 to pH 8.0. The mutants N160L and Q159L showed slightly better thermostability than wild-type PpHHDH. For most tested substrates, the two variants showed higher enantioselectivity. These findings further confirmed the importance of amino acid residues at positions 159 and 160 for the enantioselectivity of PpHHDH.

journal_name

Int J Biol Macromol

journal_title

International journal of biological macromolecules

authors

Xue F,Yu X,Shang Y,Peng C,Zhang L,Xu Q,Li A

doi

10.1016/j.ijbiomac.2019.12.203

subject

Has Abstract

pub_date

2020-03-01 00:00:00

pages

80-88

eissn

0141-8130

issn

1879-0003

pii

S0141-8130(19)37680-9

journal_volume

146

pub_type

杂志文章

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