Elsevier

Bioresource Technology

Volume 276, March 2019, Pages 154-160
Bioresource Technology

Molecular modification of a halohydrin dehalogenase for kinetic regulation to synthesize optically pure (S)-epichlorohydrin

https://doi.org/10.1016/j.biortech.2018.12.103Get rights and content

Highlights

  • Key amino acid residues located at halide ion channels of HheCPS were modified.

  • The kinetic parameters of HheCPS were regulated.

  • Optically pure (S)-ECH was biosynthesized for the first time by HheCPS mutants.

Abstract

Asymmetric synthesis of chiral epichlorohydrin (ECH) from 1,3-dichloro-2-propanol (1,3-DCP) using halohydrin dehalogenases (HHDHs) is of great value due to the 100% theoretical yield and high enantioselectivity. The vital problem in the asymmetric synthesis is to prepare optically pure ECH. In this study, key amino acid residues located at halide ion channels of HheC (P175S/W249P) (HheCPS) were modified to regulate the kinetic parameters. HheCPS I81W, F86N and V94R were constructed with the corresponding halide ion channels destroyed. The catalytically efficiencies (kcat/Km) of the three mutants exhibited 0.38-, 0.23- and 0.23-fold decrease toward (S)-ECH and the reverse reaction was significantly inhibited. As the results, (S)-ECH was synthesized with >99% enantiomeric excess (e.e.) and 63.42%, 67.08% and 57.01% yields, respectively, under 20 mM 1,3-DCP as substrate. To our knowledge, this is the first investigation of the molecule kinetic modification of HHDHs and also the first report for the biosynthesis of optically pure (S)-ECH from 1,3-DCP using HHDHs.

Introduction

Biodiesel is a biological alternative energy and boasts a huge market needs, but the huge demand leads to the accumulation of by-product glycerol with an inefficient use (Duarte et al., 2015, Shibasaki-Kitakawa et al., 2013). It is reported that 1,3-DCP which is from glycerol can be transformed into chiral ECH by a biological enzymatic method in an effective way (Xue et al., 2015a). Chiral ECH is an important versatile building block for various transformations in pharmaceutical and organic synthesis, such as β-adrenergic blocking agents, atorvastatin, L-carnitine, and trehalostatin etc. (Liu et al., 2011, Elenkov et al., 2018, Wu et al., 2010). Asymmetric synthesis of chiral ECH using HHDHs has attracted much attention because of the low catalyst cost, mild reaction conditions and 100% theoretical yield (Schallmey et al., 2013, Schallmey et al., 2014, Shen et al., 2018, Zheng et al., 2017).

HHDHs (EC 4.5.1.X) are enzymes to catalyze the reversible dehalogenation of vicinal halohydrins to form epoxides and the corresponding halide ions and H+ through a molecular kernel substitution mechanism (Hu et al., 2015, Tang et al., 2003, Xue et al., 2015b). In the presence of negative ions such as NO2, N3, OCN, HCOO, CN, SCN, Br and Cl, the reverse reaction is occurred to convert the epoxide rings into the corresponding β-substituted alcohols (Lin et al., 2011, Spelberg et al., 2001). Different approaches have been tried to prepare chiral ECH with high optical purity. It is reported that (R)-ECH was prepared with the e.e. >92.3% by HheB8 from Bradyrhizobium erythrophlei (Xue et al., 2018). In our previous work, a mutant HheCPS was constructed with high enantioselectivity and the e.e. of (S)-ECH changed greatly from 5% to 95.3%, compared with the parent HheC (Xue et al., 2015b). Recently, HheCPS was immobilized and employed in non-aqueous bioconversion system to obtain (S)-ECH with 98% e.e. and 52.34% conversion (Zhang et al., 2018). However, the optical purity of chiral ECH was not high enough (>99%) for industrial application, and as the reaction prolonged, the chiral ECH became racemic.

Due to the difference in the affinity of a HHDH to (R)- or (S)-ECH, the reverse reaction of asymmetric conversion from 1,3-DCP to chiral ECH is responsible for the conversion of (S)-ECH to 1,3-DCP and (R)-ECH, which is considered as the key factor for the low optical purity of chiral ECH products (Tang et al., 2010, Wan et al., 2018, Xue et al., 2015a). The modification of the kinetic parameters of HHDHs for the forward and reverse reactions would affect the synthesis of (S)- or (R)-ECH and thus define the optical purity of chiral ECH. Currently, most mutagenesis focused on the active sites to improve the enantioselectivity, and for preparing optically pure ECH, no report has been published about the molecular modification to alter the kinetic parameters of a HHDH for both forward and reverse reactions.

In the present study, the amino acid residues located at the halide ion channels of HheCPS were predicted by specialized toolkit MOLE 2.0 and site-directed saturation mutagenesis was carried out to modify the kinetic characters of the enzyme. The positive mutants were employed for the synthesis of (S)-ECH with >99% e.e. To our knowledge, this is the first investigation of the molecule kinetic modification of a HHDH and also the first report for the biosynthesis of optically pure (S)-ECH from 1,3-DCP using HHDH as catalyst.

Section snippets

Materials

1,3-DCP, (S)-ECH and (R)-ECH were purchased from J & K Scientific Ltd. (Shanghai, China). Phanta Max Super-Fidelity DNA Polymerase and DpnI methylation-sensitive restriction enzyme were purchased from Vazyme Biotech Co., Ltd. (Nanjing, China). AxyPrep Plasmid Miniprep Kit was purchased from Axygene Biotech Ltd. (Hangzhou, China). Kanamycin (Kan) and the inducer isopropyl β-d-thiogalactoside (IPTG) were purchased from Sango Biotech Co., Ltd. (Shanghai, China). PCR cleanup kit was purchased from

Homology modeling and docking

HheCPS with high enantioselectivity was employed as the parent enzyme for molecular modification in the present study. The 3-D structure model for HheCPS was generated using Modeller 9.19 program according to HheC crystal structure (PDB ID: 1ZO8) and was evaluated by Procheck (de Jong et al., 2002). The simulated model with the highest GA341 score and the lowest DOPE score was selected for further research. Halide ion channels of the protein were predicted by MOLE 2.0, the probe radius was set

Conclusions

In our present study, using the molecule kinetic to regulate HheCPS mutants, optically pure (S)-ECH (e.e. >99%) was firstly synthesized. Our study provides a class of HHDH catalysts for the preparation of chiral ECH with high optical purity and considerable yield. Our present work makes contribution to the development of green technology for the synthesis of chiral ECH. However, more works need to be done including protein engineering, chemical modification of HHDHs and reaction process control

Acknowledgments

This work was financially supported by the Public Welfare Technology Application Research Project of Zhejiang Province (No. 2016C31013), the Natural Science Foundation of Zhejiang Province (No. LQ14B060004), the Project from Department of Education of Zhejiang Province (No. Y201534805), and the Preferred Fund of Postdoctoral Scientific Research Project of Zhejiang (No. BSH1602103).

Conflict of interest

The authors declare that they have no conflict of interest.

References (36)

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