Preparation of (S)-epichlorohydrin using a novel halohydrin dehalogenase by selective conformation adjustment.

Chiral epichlorohydrin (ECH) is an attractive intermediate for chiral pharmaceuticals and chemicals preparation. The asymmetric synthesis of chiral ECH using 1,3-dicholoro-2-propanol (1,3-DCP) catalyzed by a haloalcohol dehalogenase (HHDH) was considered as a feasible approach. However, the reverse ring opening reaction caused low optical purity of chiral ECH, thus severely restricts the industrial application of HHDHs. In the present study, a novel selective conformation adjustment strategy was developed with an engineered HheCPS to regulate the kinetic parameters of the forward and reverse reactions, based on site saturation mutation and molecular simulation analysis. The HheCPS mutant E85P was constructed with a markable change in the conformation of (S)-ECH in the substrate pocket and a slight impact on the interaction between 1,3-DCP and the enzyme, which resulted in the kinetic deceleration of the reverse reactions. Compared with HheCPS, the catalytic efficiency (kcat(S)-ECH/Km(S)-ECH) of the reversed reaction dropped to 0.23-fold (from 0.13 to 0.03 mM-1 s-1), while the catalytic efficiency (kcat(1,3-DCP)/Km(1,3-DCP)) of the forward reaction only reduced from 0.83 to 0.71 mM-1 s-1. With 40 mM 1,3-DCP as substrate, HheCPS E85P catalyzed the synthesis of (S)-ECH with the yield up to 55.35% and the e.e. increased from 92.54 to >99%. Our work provided an effective approach for understanding the stereoselective catalytic mechanism as well as the green manufacturing of chiral epoxides.

Infrared Spectrum Characteristics and Quantification of OH Groups in Coal.

The KBr pellet press method for detecting the infrared spectrum of coal is one of the commonly used methods for analyzing the types and content of functional groups in coal. However, KBr crystalline water or moisture has a significant impact on the peak position, peak shape, and peak area of the organic O-H based stretching vibration wave in coal. In this paper, the theoretical characteristics of infrared spectra of phenols and alcohols have been simulated and analyzed using the Gaussian 16 series of programs. Four infrared spectral analysis techniques, in situ infrared, KBr pellet press, dry KBr pellet press, and paste methods, have been used to detect the infrared spectra of coal. The results show that the stretching vibration peaks of free O-H radicals without hydrogen bonding are located between 3700 and 3600 cm-1. After the O-H form hydrogen bonds with each other, the O-H stretching vibration frequency moves toward the low frequency direction, and the lower the wavenumber, the more O-H content. The conventional KBr gasket manufacturing process will absorb moisture in the air to interfere with the hydroxyl absorption peak of coal, and the experimental process requires absolute drying. The relative content of hydroxyl in coal can be compared and analyzed based on the peak position, peak shape, and peak area of the hydroxyl stretching vibration wave. Quantitative analysis of hydroxyl groups in coal also requires combination of elemental analysis and X-ray photoelectron spectroscopy.

Engineering the Active Site Lid Dynamics to Improve the Catalytic Efficiency of Yeast Cytosine Deaminase.

Conformational dynamics is important for enzyme catalysis. However, engineering dynamics to achieve a higher catalytic efficiency is still challenging. In this work, we develop a new strategy to improve the activity of yeast cytosine deaminase (yCD) by engineering its conformational dynamics. Specifically, we increase the dynamics of the yCD C-terminal helix, an active site lid that controls the product release. The C-terminal is extended by a dynamical single α-helix (SAH), which improves the product release rate by up to ~8-fold, and the overall catalytic rate kcat by up to ~2-fold. It is also shown that the kcat increase is due to the favorable activation entropy change. The NMR H/D exchange data indicate that the conformational dynamics of the transition state analog complex increases as the helix is extended, elucidating the origin of the enhanced catalytic entropy. This study highlights a novel dynamics engineering strategy that can accelerate the overall catalysis through the entropy-driven mechanism.

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

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.

Biosynthesis of chiral epichlorohydrin using an immobilized halohydrin dehalogenase in aqueous and non-aqueous phase.

Asymmetric synthesis of chiral epichlorohydrin (ECH) from 1,3-dichloro-2-propanol (1,3-DCP) using halohydrin dehalogenase (HHDH) is of great value due to the 100% theoretical yield and high enantioselectivity. In this study, HheC (P175S/W249P) was immobilized on an A502Ps resin and used for the preparation of (S)-ECH. In aqueous system, the immobilized HheC catalyzed the biosynthesis of (S)-ECH with 83.78% yield and 92.53% enantiomeric excess (ee) at 1,3-DCP concentration of 20 mM. The non-aqueous system was further developed using water saturated ethyl acetate as solvent and reaction phase. The non-aqueous bioconversion system showed higher enantioselectivity (>98% ee) toward (S)-ECH production with modest conversion (52.34%) compared with ever reported aqueous reactions. Batch reactions were performed in a packed-bed bioreactor for 45 batches in aqueous phase and 24 batches in non-aqueous phase. The present work demonstrated the potential of immobilized HheC (P175S/W249P) in aqueous and non-aqueous phase biosynthesis of chiral ECH.