Halohydrin Dehalogenase Research Articles

Heterologous overexpression of Pseudomonas umsongensis halohydrin dehalogenase in Escherichia coli and its application in epoxide asymmetric ring opening reactions

Abstract A new halohydrin dehalogenase-like gene (HHDH) in the genome of Pseudomonas umsongensis YCIT1612 was identified, cloned and over-expressed in Escherichia coli. HHDH-Pu exhibited the highest sequence identity of 45% in comparison with the previously reported HHDHs. The recombinant HHDH-Pu was purified to homogeneity and its catalytic characteristics in epoxide ring opening was explored. Compared to other reported HHDHs, HHDH-Pu showed higher catalytic activity, exhibiting varied activities (2.4–50.4 U/mg) toward all the tested epoxides. It catalyzed the ring opening of epoxides with low to moderate enantioselectivity (E=1.13-10.89). Rac-PGE was found to be the most selective among the tested epoxides. In the ring opening of rac-PGE, nucleophiles such as Cl−, N3−, NO2−, Br− or SCN− were accepted. N3− was selected as the optimal nucleophile. Some parameters that may alter this bio-resolution, such as the concentration of N3−, temperature and pH were evaluated. By optimizing the reaction conditions, the residual (S)-PGE could be obtained in 23.5% yield and more than 99% ee from 40 mM rac-PGE.

Identification and characterization of a highly S-enantioselective halohydrin dehalogenase from Tsukamurella sp. 1534 for kinetic resolution of halohydrins

Halohydrin dehalogenases are remarkable enzymes which possess promiscuous catalytic activity and serve as potential biocatalysts for the synthesis of chiral halohydrins, epoxides and β-substituted alcohols. The enzyme HheC exhibits a highly R enantioselectivity in the processes of dehalogenation of vicinal halohydrins and ring-opening of epoxides, which attracts more attentions in organic synthesis. Recently dozens of novel potential halohydrin dehalogenases have been identified by gene mining, however, most of the characterized enzymes showed low stereoselectivity. In this study, a novel halohydrin dehalogenase of HheA10 from Tsukamurella sp. 1534 has been heterologously expressed, purified and characterized. Substrate spectrum and kinetic resolution studies indicated the HheA10 was a highly S enantioselective enzyme toward several halohydrins, which produced the corresponding epoxides with the ee (enantiomeric excess) and E values up to >99% and >200 respectively. Our results revealed the HheA10 was a promising biocatalyst for the synthesis of enantiopure aromatic halohydrins and epoxides via enzymatic kinetic resolution of racemic halohydrins. What’s more important, the HheA10 as the first individual halohydrin dehalogenase with the highly S enantioselectivity provides a complementary enantioselectivity to the HheC.

Micromechanics of Anisotropic Cross-Linked Enzyme Crystals

Catalytic activity of protein crystals correlates with their particle size due to diffusion limitations. However, because the particles need to be restrained during, e.g., a filtration process, keeping a minimum particle size is required. Thus, knowledge of mechanical properties of enzyme crystals is needed for downstream process design in the biopharmaceutical industry to avoid particle breakage. In this study, hardness and Young’s modulus of cross-linked lysozyme crystals and cross-linked halohydrin dehalogenase from Ilumatobacter coccineus (HheG) crystals are evaluated using atomic force microscopy. The results show that hardness of lysozyme and HheG crystals is in the range of 2–22 MPa. For Young’s modulus of lysozyme crystals, values between 40 and 1820 MPa are measured, while HheG crystals ranging between 40 and 1200 MPa, respectively. The results for lysozyme crystals are comparable to those published in the current literature on native protein crystals. Hence, the cross-linking seems not to signif...

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.

Identification and Characterization of a Novel Halohydrin Dehalogenase from Bradyrhizobium erythrophlei and Its Performance in Preparation of Both Enantiomers of Epichlorohydrin

The new halohydrin dehalogenase-like gene (HheB8) in the genome of Bradyrhizobium erythrophlei was identified, synthesized and expressed in Escherichia coli. The encoded protein (HheB8) was purified to homogeneity as a molecular mass of 26 kDa and characterized using the model substrate of 1,3-dicholoro-2-propanol (1,3-DCP). The purified HheB8 was optimally active at pH 9.5 and 45 °C, and highly stable at pH 6.0–8.0 and 40 °C or below. The enzyme displayed a very broad substrate specificity, exhibiting good enantioselectivity for the transformation of prochiral 1,3-DCP and 1,3-dibromo-2-propanol to (R)-ECH (92.3% enantiomeric excess ee) and (R)-epibromohydrin (81.3% ee). For all tested epoxides, except styrene oxide, the (R)-enantiomer was converted first, affording (S)-isomer in 99% ee. Furthermore, the potential for using a single HHDH to enantioselectively produce both enantiomers of ECH was analyzed. The bioconversion process for production of (R)-ECH from 1,3-DCP by the recombinant HheB8 was developed and a maximum ee of 93.4% was achieved. Kinetically resolved racemic ECH (30 mM) to provide (S)-ECH with 99% ee and 33.4% yield. To the best of our knowledge, this is the first report of HheB8 used as biocatalyst in the production of enantiopure ECH. Therefore, this recombinant HHDH could be a potential candidate for application in the synthesis of chiral chemicals from prochiral halohydrins and racemic epoxides.

Covalent immobilization of halohydrin dehalogenase for efficient synthesis of epichlorohydrin in an integrated bioreactor.

Halohydrin dehalogenase (HHDH)-mediated dehalogenation of 1,3-dichloro-2-propanol (1,3-DCP) is a key step in the chemoenzymatic synthesis of epichlorohydrin (ECH) from glycerol. In this study, a covalent immobilization strategy was employed to enhance the stability of Agrobacterium tumefaciens HHDH using epoxy resin ES-103B as a carrier. Under optimal conditions, the activity recovery of ES-103B-immobilized HHDH (HHDH@ES-103B) was 62.4% and the specific activity was 1604 U/g. The HHDH@ES-103B exhibited excellent thermostability, with a half-life of 68.6 days at 40°C, which is 8.0-times higher than that of the free HHDH. A semicontinuous biotransformation of 1,3-DCP to ECH was performed using HHDH@ES-103B as biocatalyst in a recirculating packed bed reactor (RPBR), resulting in an ECH yield of 94.2%, with an average productivity of 5.2 g/L/h. The RPBR system exhibited a high operational stability and even after 50 cycles of reaction, it retained > 90% of the initial conversion. Furthermore, an integrated bioprocess based on in situ product recovery (ISPR) was developed in RPBR to overcome product inhibition. The integrated bioreactor equipped with an external macroporous adsorption resin HZD-9 column led to another 1.6-fold increase in ECH productivity to 8.46 g/L/h. This improved stability and reusability of HHDH@ES-103B demonstrated its potential for the biotransformation of 1,3-DCP to ECH. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:784-792, 2018.

Cross-linked enzyme aggregates (CLEAs) of halohydrin dehalogenase from Agrobacterium radiobacter AD1: Preparation, characterization and application as a biocatalyst

Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) shows great potential to produce valuable optically pure epoxides and β-substituted alcohols. However, this enzyme has been reported to be very sensitive and less stable under oxidative conditions. Enzyme immobilization represents a powerful means to overcome this limitation and provides the enzyme characteristics of a biocatalyst. In this study, the crude extract of HheC was directly subjected to enzyme immobilization using a carrier-free cross-linked enzyme aggregates (CLEAs) method. The results showed that under the optimized conditions, the obtained HheCCLEAs retained more than 90% activity of the free enzyme; preserved more than 50% of their original activity after storage at 4 °C for 2 months, even in the absence of a reducing agent; displayed a strong tolerance to organic solvents with fully active after incubation in the presence of 50% cyclohexane and n-hexane for 5 h; the presence of organic solvents could minimize the negative effect of enzyme immobilization on the enzntioselectivity of HheC. Most importantly, HheCCLEAs maintained more than 70% activity after 10 reusability cycles. The utility of HheCCLEAs as a valuable biocatalyst was exhibited by the kinetic resolution of azide-mediated ring-opening reaction of rac-1,2-epoxy-2-methylbutane. These results indicated that HheCCLEAs overcame some disadvantages of free enzymes to become biocatalysts. Together with further engineering of the enzyme, HheCCLEAs could become a promising biocatalyst for the synthesis of valuable chiral compounds.

Halohydrin dehalogenase-catalysed transformations of epifluorohydrin

Biocatalytic ring-opening of epifluorohydrin has been performed by using halohydrin dehalogenase. The enzyme from Mycobacterium sp. GP1 (HheB2) catalysed reaction with high regioselectivity and low enantioselectivity in the presence of different nucleophiles, producing racemic 1-substituted 3-fluoro-2-propanols. No by-products resulting from the ring-closure reaction have been detected, confirming that vicinal fluoro alcohols are not substrates for HHDH. Four different 3-fluoro-2-propanols were prepared under mild reaction conditions starting from epifluorohydrin. High conversions of 85–100% were reached within 1–3 h and depending on the nucleophile used products were isolated in 31–92% yields.

HheG, a Halohydrin Dehalogenase with Activity on Cyclic Epoxides

Halohydrin dehalogenases (HHDHs) are of biotechnological interest due to their promiscuous epoxide ring-opening activity with a set of negatively charged nucleophiles, enabling the formation of C–C, C–N, or C–O bonds. The recent discovery of HHDH-specific sequence motifs aided the identification of a large number of halohydrin dehalogenases from public sequence databases, enlarging the biocatalytic toolbox substantially. During the characterization of 17 representatives of these phylogenetically diverse enzymes, one HHDH, namely HheG from Ilumatobacter coccineus, was identified to convert cyclic epoxide substrates. The enzyme exhibits significant activity in the azidolysis of cyclohexene oxide and limonene oxide with turnover numbers of 7.8 and 44 s–1, respectively. As observed for other HHDHs, the cyanide-mediated epoxide ring-opening proceeded with lower rates. Wild-type HheG displays modest enantioselectivity, as the resulting azido- and cyanoalcohols of cyclohexene oxide ring-opening were obtained in ...

Development of an Enzymatic Process for the Production of (R)-2-Butyl-2-ethyloxirane

An epoxide resolution process was rapidly developed that allowed access to multigram scale quantities of (R)-2-butyl-2-ethyloxirane 2 at greater than 300 g/L reaction concentration using an easy-to-handle and store lyophilized powder of epoxide hydrolase from Agromyces mediolanus. The enzyme was successfully fermented on a 35 L scale and stability increased by downstream processing. Halohydrin dehalogenases also gave highly enantioselective resolution but were shown to favor hydrolysis of the (R)-2 epoxide, whereas epoxide hydrolase from Aspergillus niger instead provided (R)-7 via an unoptimized, enantioconvergent process.

Effect of Zn2+ on halohydrin dehalogenase expression and accumulation through multi-parameter correlation research with Escherichia coli P84A/MC1061

ABSTRACT Using 5 Zn2+ supplementation strategies in a 50 L batch bioreactor named FUS-50L(A), possible correlations among Zn2+ content and addition timing, physiologic activity (PA), halohydrin dehalogenase (HheC) accumulation of Escherichia coli P84A/MC1061 were systematically investigated. First, Zn2+ was confirmed as the significant factor, and its optimal concentration for HheC expression was 3.87 mg/L through fermentation experiments in shaking flasks. Second, based on experimental results from the different strategies, it was found that PA, nutrient consumption rate (NCR) and specific growth rate (μ) for E. coli P84A/MC1061 were promoted in the log phase (4–8 h) under appropriate Zn2+ concentrations in the lag phase and late log phase. Furthermore cell biomass was also increased to a higher level and the maximum HheC activity (i.e. HheCmax) was increased by 9.80%, and the time to reach HheCmax was reduced from 16 to 12 hours. Furthermore, appropriate supplementation of Zn2+ caused higher μ for E. coli P84A/MC1061, which resulted in more rapid accumulation of increased acetic acid concentrations, leading to higher acetic acid consumption avoiding any negative effects on producing HheC because of carbon source being exhausted prematurely and acetic acid being consumed rapidly.

Enhancing the biocatalytic manufacture of the key intermediate of atorvastatin by focused directed evolution of halohydrin dehalogenase

Halohydrin dehalogenases (HHDHs) are biocatalytically interesting enzymes due to their ability to form C-C, C-N, C-O, and C-S bonds. One of most important application of HHDH was the protein engineering of HheC (halohydrin dehalogenase from Agrobacterium radiobacter AD1) for the industrial manufacturing of ethyl (R)-4-cyano-3-hydroxybutanoate (HN), a key chiral synthon of a cholesterol-lowering drug of atorvastatin. During our development of an alternative, more efficient and economic route for chemo-enzymatic preparation of the intermediate of atorvastatin, we found that the HheC2360 previously reported for HN manufacture, had insufficient activity for the cyanolysis production of tert-butyl (3 R,5 S)-6-cyano-3,5-dihydroxyhexanoate (A7). Herein, we present the focused directed evolution of HheC2360 with higher activity and enhanced biocatalytic performance using active site mutagenesis. Through docking of the product, A7, into the crystal structure of HheC2360, 6 residues was selected for combined active sites testing (CASTing). After library screening, the variant V84G/W86F was identified to have a 15- fold increase in activity. Time course analysis of the cyanolysis reaction catalyzed by this variant, showed 2- fold increase in space time productivity compared with HheC2360. These results demonstrate the applicability of the variant V84G/W86F as a biocatalyst for the efficient and practical production of atorvastatin intermediate.

Exploring the thermostable properties of halohydrin dehalogenase from Agrobacterium radiobacter AD1 by a combinatorial directed evolution strategy.

As a crucial factor for biocatalysts, protein thermostability often arises from a combination of factors that are often difficult to rationalize. In this work, the thermostable nature of halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) was systematically explored using a combinatorial directed evolution approach. For this, a mutagenesis library of HheC mutants was first constructed using error-prone PCR with low mutagenesis frequency. After screening approximately 2000 colonies, six mutants with eight mutation sites were obtained. Those mutation sites were subsequently combined by adopting several rounds of iterative saturation mutagenesis (ISM) approach. After four rounds of saturation mutagenesis, one best mutant ISM-4 with a 3400-fold improvement in half-life (t 1/2) inactivation at 65°C, 18°C increase in apparent T m value, and 20°C increase in optimum temperature was obtained, compared to wild-type HheC. To the best of our knowledge, the mutant represents the most thermostable HheC variant reported up to now. Moreover, the mutant was as active as wild-type enzyme for the substrate 1,3-dichloro-2-propanol, and they remained most enantioselectivity of wild-type enzyme in the kinetic resolution of rac-2-chloro-1-phenolethanol, exhibiting a great potential for industrial applications. Our structural investigation highlights that surface loop regions are hot spots for modulating the thermostability of HheC, the residues located at these regions contribute to the thermostability of HheC in a cooperative way, and protein rigidity and oligomeric interface connections contribute to the thermostability of HheC. All of these essential factors could be used for further design of an even more thermostable HheC, which, in turn, could greatly facilitate the application of the enzyme as a biocatalyst.

A robust cosolvent-compatible halohydrin dehalogenase by computational library design.

To improve the applicability of halohydrin dehalogenase as a catalyst for reactions in the presence of organic cosolvents, we explored a computational library design strategy (Framework for Rapid Enzyme Stabilization by Computational libraries) that involves discovery and in silico evaluation of stabilizing mutations. Energy calculations, disulfide bond predictions and molecular dynamics simulations identified 218 point mutations and 35 disulfide bonds with predicted stabilizing effects. Experiments confirmed 29 stabilizing point mutations, most of which were located in two distinct regions, whereas introduction of disulfide bonds was not effective. Combining the best mutations resulted in a 12-fold mutant (HheC-H12) with a 28°C higher apparent melting temperature and a remarkable increase in resistance to cosolvents. This variant also showed a higher optimum temperature for catalysis while activity at low temperature was preserved. Mutant H12 was used as a template for the introduction of mutations that enhance enantioselectivity or activity. Crystal structures showed that the structural changes in the H12 mutant mostly agreed with the computational predictions and that the enhanced stability was mainly due to mutations that redistributed surface charges and improved interactions between subunits, the latter including better interactions of water molecules at the subunit interfaces.

Azidolysis of epoxides catalysed by the halohydrin dehalogenase from Arthrobacter sp. AD2 and a mutant with enhanced enantioselectivity: an (S)-selective HHDH

Halohydrin dehalogenase from Arthrobacter sp. AD2 catalysed azidolysis of epoxides with high regioselectivity and low to moderate (S)-enantioselectivity (E=1–16). Mutation of the asparagine 178 to alanine (N178A) showed increased enantioselectivity towards styrene oxide derivatives and glycidyl ethers. Conversion of aromatic epoxides was catalysed by HheA-N178A with complete enantioselectivity, however the regioselectivity was reduced. As a result of the enzyme-catalysed reaction, enantiomerically pure (S)-β-azido alcohols and (R)-α-azido alcohols (ee⩾99%) were obtained.

Recent advances on halohydrin dehalogenases-from enzyme identification to novel biocatalytic applications.

Halohydrin dehalogenases are industrially relevant enzymes that catalyze the reversible dehalogenation of vicinal haloalcohols with formation of the corresponding epoxides. In the reverse reaction, also other negatively charged nucleophiles such as azide, cyanide, or nitrite are accepted besides halides to open the epoxide ring. Thus, novel C-N, C-C, or C-O bonds can be formed by halohydrin dehalogenases, which makes them attractive biocatalysts for the production of various β-substituted alcohols. Despite the fact that only five individual halohydrin dehalogenase enzyme sequences have been known until recently enabling their heterologous production, a large number of different biocatalytic applications have been reported using these enzymes. The recent characterization of specific sequence motifs has facilitated the identification of novel halohydrin dehalogenase sequences available in public databases and has largely increased the number of recombinantly available enzymes. These will help to extend the biocatalytic repertoire of this enzyme family and to foster novel biotechnological applications and developments in the future. This review gives a general overview on the halohydrin dehalogenase enzyme family and their biochemical properties and further focuses on recent developments in halohydrin dehalogenase biocatalysis and protein engineering.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7750-y) contains supplementary material, which is available to authorized users.

Biochemical and biocatalytic characterization of 17 novel halohydrin dehalogenases.

Halohydrin dehalogenases are rare but catalytically remarkable enzymes since they are able to form novel C-C, C-O, C-N, or C-S bonds. Very recently, a motif-based sequence database mining approach resulted in the identification of 37 novel halohydrin dehalogenase enzymes, many of them exhibiting only low sequence similarity to previously known halohydrin dehalogenases. In an attempt to explore the biocatalytic potential of these newly identified enzymes, 17 representatives from all six phylogenetic subtypes were heterologously produced in Escherichia coli, purified and characterized to determine their substrate scopes in the dehalogenation and epoxide ring-opening reaction. Several enzymes with broad substrate spectra were identified exhibiting high activities towards a selection of typical substrates. Moreover, four halohydrin dehalogenases were found to be significantly more thermostable than thepreviously known HheC from Agrobacterium radiobacter AD1. Investigation of the enzymes' stereoselectivity in the dehalogenation of racemic 2-chloro-1-phenylethanol revealed that their stereopreference correlates with the phylogenetic placing of the enzymes in subtypes A through G. Furthermore, the biocatalytic potential of these novel halohydrin dehalogenases was investigated in the preparation of ethyl 4-cyano-3-hydroxybutyrate, a statin side-chain precursor. Though none of the active enzymes selectively formed the required (R)-enantiomer, several halohydrin dehalogenases were identified with significantly higher activity in the conversion compared to HheC, making them promising candidates for this industrially relevant reaction.

Synthesis aided design: The biological design-build-test engineering paradigm?

Synthesis aided design: The biological design-build-test engineering paradigm?

Efficient synthesis of (S)-epichlorohydrin in high yield by cascade biocatalysis with halohydrin dehalogenase and epoxide hydrolase mutants

Enantioselective biotransformation of prochiral 1,3-DCP by halohydrin dehalogenases (HHDHs) is particularly attractive since 100% yield of chiral epichlorohydrin (ECH) may be obtained. HheC mutant (P175S/W249P) displayed greatly improved enantiomeric excess (ee) of (S)-ECH from 5% to 95.3% in the catalyzed dehalogenation of 1,3-DCP at pH8.0. (S)-ECH was enantioselectively biotransformed from 40mM 1,3-DCP with 92.3% ee and 93.2% yield at pH10.0. To increase the ee of (S)-ECH, the catalysis was carried out using HheC mutant coupled with epoxide hydrolase mutant and the maximum yield and ee of (S)-ECH reached 91.2% and >99%.

ChemInform Abstract: Oxazolidinone Synthesis Through Halohydrin Dehalogenase‐Catalyzed Dynamic Kinetic Resolution.

An efficient dynamic kinetic resolution protocol using a single enzyme is described. Both the kinetic resolution and substrate racemization are catalyzed by halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC). The HheC-catalyzed reaction of epibromohydrin and 2-bromomethyl-2-methyloxirane with sodium cyanate afforded 5-substituted 2-oxazolidinones in high yields (97% and 87%) and high optical purity (89% and > 99% ee) in the presence of catalytic amounts of bromide ion. These compounds are valuable building blocks with diverse synthetic applications.