Nitrite‐mediated synthesis of chiral epichlorohydrin using halohydrin dehalogenase from Agrobacterium radiobacter AD1

Abstract

In the current study, the haloalcohol dehalogenase HheC gene from Agrobacterium radiobacter AD1 was synthesized and expressed in Escherichia coli. After purification using Ni-nitrilotriacetic acid affinity chromatography, HheC was used in the synthesis of chiral epichlorohydrin in the presence of NO₂⁻. The optimal pH, temperature, and NO₂⁻ concentration for enantioselectivity are 5.0, 37°C, and 60 mM, respectively. The maximum velocity and Michaelis constant values for (S)-epichlorohydrin are 714.3 µmol min⁻¹ mg⁻¹ and 17.2 mM, respectively, whereas those for (R)-epichlorohydrin are 166.8 µmol min⁻¹ mg⁻¹ and 29.0 mM, respectively. Under optimal conditions, (R)-epichlorohydrin with 99% enantiomeric excess was obtained after an 18 Min reaction; the yield reached 41%, which is the highest amount obtained for chiral epichlorohydrin synthesis using haloalcohol dehalogenase. In addition, (R)-epichlorohydrin with 99% enantiomeric excess was successfully obtained from 1,3-dichloro-2-propanol by the ring opening of racemic epichlorohydrin in the presence of NO₂⁻ after the ring closure of 1,3-dichloro-2-propanol with HheC. To the best of our knowledge, the current study is the first report on the kinetic resolution of epichlorohydrin with NO₂⁻ and synthesis of chiral epichlorohydrin with 99% enantiomeric excess from 1,3-dichloro-2-propanol by combining ring closure of 1,3-dichloro-2-propanol and ring opening of racemic epichlorohydrin.

Investigation of Halohydrins Degradation by Whole Cells and Cell-free Extract of Pseudomonas putida DSM 437: A Kinetic Approach

“…This indicates that ECH is by far the most potent inhibitor of 3-CPD degradation by the cell-free extract of P. putida DSM 437. Inhibition of the enzyme (a halohydrin dehalogenase) catalyzing the transformation of 1,3-DCP to ECH has been confirmed by previous studies [29][30] . IC 50 is the concentration of an inhibitor required to reduce the rate of an enzymatic reaction by 50 %.…”

Chemoenzymatic preparation of (S)-p-nitrostyrene oxide from p-nitrophenacyl bromide by recombinant Escherichia coli cells expressing a novel halohydrin dehalogenase

“…However, the high conversion of BNPE and yield of pNSO were considered as an advantage for the next bioresolution of rac-pNSO. The conversion (100%) and yield (91.1%) was also found to be the highest level for the ring-closure reaction among the known EHs [13,17]. Thereafter, the bioresolution of rac-pNSO catalyzed by whole cells of E. coli/Syhhdh with NO 2 − was performed just by changing the reaction conditions.…”

“…The resultant recombinant plasmid, pET-28a(+)-Syhhdh, was then transformed into E. coli BL21(DE3). Expression of reSyHhdh was performed according to the method described previously [17]. The E. coli/Syhhdh cells induced by IPTG were harvested by centrifugation, then resuspended in 20 mM Tris-HCl buffer (pH 7.9) containing 500 mM NaCl, disrupted by sonication and centrifuged to remove cell debris.…”

Expanding the Halohydrin Dehalogenase Enzyme Family: Identification of Novel Enzymes by Database Mining

“…Besides being useful for the production of enantiopure haloalcohols (4-6) and epoxides (5,(7)(8)(9), these enzymes can also be applied in the formation of novel carbon-carbon, carbon-nitrogen, or carbon-oxygen bonds. Due to the promiscuous epoxide ring-opening activity of these enzymes, cyanide, azide, and nitrite, for example, are accepted as nucleophiles in the ring-opening reaction, leading to a diverse range of products (10).…”

“…Due to the promiscuous epoxide ring-opening activity of these enzymes, cyanide, azide, and nitrite, for example, are accepted as nucleophiles in the ring-opening reaction, leading to a diverse range of products (10). Examples of important HHDH applications are the production of optically pure C 3 or C 4 fine-chemical precursors (5,8,11,12), including the multiton-scale production of enantiopure (R)-4-cyano-3-hydroxybutyrate esters for statin drugs (13), and the production of chiral tertiary alcohols (14)(15)(16)(17), for which conventional organic synthesis is rather challenging (Fig. 1) (18,19).…”