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Halohydrin dehalogenases (HHDHs) have demonstrated significant potential as biocatalysts for synthesizing industrial heterocyclic compounds and chiral alkanolamines from aromatic substrates with NaOCN that are not feasible through chemosynthesis. To further study the HHDH reaction in real industrial applications, a diverse panel of HHDHs has been screened for their ability to convert aliphatic cyclohexene oxide. By using the best HHDH variant in form of a whole cell biocatalyst, a novel chemical process, regulated by the cascaded chemoenzymatic reactions for pilot‐scale ring‐opening reactions, has been successfully developed. Using the best HHDH hit in whole‐cell form provides a viable bio‐catalytic approach for the synthesis of related fine chemicals. Our research provides a promising strategy for scaling up non‐native ring‐opening reactions from the laboratory to industrial applications. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We described the protein engineering of the halohydrin dehalogenase HheG to enhance its R enantioselectivity for the synthesis of chiral 4‐aryloxazolidinones via α‐regioselective ring‐opening of aryl epoxides with cyanate. Additionally, we achieved the inversion of its regioselectivity and enantioselectivity, resulting in a HheG variant tailored for the synthesis of chiral 5‐aryloxazolidinones via β‐regioselective and S‐enantioselective ring‐opening of aryl epoxides with cyanate. Leveraging these engineered mutants, we developed a biocatalytic platform capable of synthesis of both chiral 4‐aryloxazolidinones (up to 47% yield, 98% ee, and 99% regioselectivity) and chiral 5‐aryloxazolidinones (up to 46% yield, >99% ee, and 98% regioselectivity). Furthermore, a collaborative approach utilizing two regio‐ and enantioselective HheG mutants has been demonstrated to enable the simultaneous synthesis of chiral 4‐aryloxazolidinones and chiral 5‐aryloxazolidinones. [ABSTRACT FROM AUTHOR]

In this study, a lyophilization protocol was established for storable and ready-to-use halohydrin dehalogenase (HHDH) from Agrobacterium radiobacter AD1 (HheC), as a model enzyme. HheC showed high activity preservation after the lyophilization procedure, even in the absence of lyoprotectants. Freeze-dried HheC exhibited higher residual activity compared to the liquid form when stored at 50 °C. Among tested additives the formulation with glycine was found to be the best choice regarding the mechanical strength of the lyophilized pellets. Besides, the results revealed significant stability of dry HheC exposed to MTBE, toluene, and 2-MeTHF. However, no activity of the lyophilized HheC was observed in the dehalogenation of 1,3-dichloro-2-propanol (1,3-DCP) in non-aqueous organic media. This study showed that the lyophilization method provides a convenient way for long-term storage of HHDH enzymes and could have an impact on their wider application in the research and industrial biotechnology. [Display omitted] • Freeze-drying of Agrobacterium radiobacter (HheC) halohydrin dehalogenase was studied. • High HheC resilience towards freeze-drying stress was established. • Long-term storage of HheC is provided by freeze-drying. • Glycine and Na 2 HPO 4 were the most effective additives. • Lyophilized HheC shows high resistance to elevated temperatures and organic solvents. [ABSTRACT FROM AUTHOR]

Halohydrin dehalogenase HheG is an industrially interesting biocatalyst for the preparation of different β‐substituted alcohols starting from bulky internal epoxides. We previously demonstrated that the immobilization of different HheG variants in the form of cross‐linked enzyme crystals (CLECs) yielded stable and reusable enzyme immobilizes with increased resistance regarding temperature, pH, and the presence of organic solvents. Now, to further establish their preparative applicability, HheG D114C CLECs cross‐linked with bis‐maleimidoethane have been successfully produced on a larger scale using a stirred crystallization approach, and their application in different chemical reactor types (stirred tank reactor, fluidized bed reactor, and packed bed reactor) was systematically studied and compared for the ring opening of cyclohexene oxide with azide. This revealed the highest obtained space‐time yield of 23.9 kgproduct gCLEC−1 h−1 Lreactor volume−1 along with the highest achieved product enantiomeric excess [64%] for application in a packed‐bed reactor. Additionally, lyophilization of those CLECs yielded a storage‐stable HheG preparation that still retained 67% of initial activity (after lyophilization) after 6 months of storage at room temperature. [ABSTRACT FROM AUTHOR]

Biocatalytic transformations in organic synthesis often require the use of organic solvents to improve substrate solubility and promote the product formation. Halohydrin dehalogenases (HHDHs) are enzymes that catalyze the formation and conversion of epoxides, important synthetic class of compounds that are often sparingly soluble in water and prone to hydrolysis. In this study, the activity, stability, and enantioselectivity of HHDH from Agrobacterium radiobacter AD1 (HheC) in form of cell-free extract were evaluated in various aqueous-organic media. A correlation was discovered between the enzyme activity in the ring-closure reaction and logP of the solvent. Knowledge of such a relationship makes biocatalysis with organic solvents more predictable, which may reduce the need to experiment with a variety of solvents in the future. The results revealed a high enzyme compatibility with hydrophobic solvents (e.g., n-heptane) in terms of activity and stability. Regarding the HHDH applicability in an organic medium, inhibitions by a number of solvents (e.g., THF, toluene, chloroform) proved to be a more challenging problem than the protein stability, especially in the ring-opening reaction, thus suggesting which solvents should be avoided. In addition, solvent tolerance of the thermostable variant ISM-4 was also evaluated, revealing increased stability and to a lesser extent enantioselectivity compared to the wild-type. This is the first time such a systematic analysis has been reported, giving insight into the behavior of HHDHs in nonconventional media and opening new opportunities for the future biocatalytic applications. Key points: • HheC performs better in the presence of hydrophobic than hydrophilic solvents. • Enzyme activity in the PNSHH ring-closure reaction is a function of the logP. • Thermostability of ISM-4 variant is accompanied by superior solvent tolerance. [ABSTRACT FROM AUTHOR]

Expanding the enzymatic toolbox for the green synthesis of valuable molecules is still of high interest in synthetic chemistry and the pharmaceutical industry. Chiral thiiranes are valuable sulfur‐containing heterocyclic compounds, but relevant methods for their enantioselective synthesis are limited. Herein, we report a biocatalytic thionation strategy for the enantioselective synthesis of thiiranes, which was developed based on the halohydrin dehalogenase (HHDH)‐catalyzed enantioselective ring‐opening reaction of epoxides with thiocyanate and a subsequent nonenzymatic rearrangement process. A novel HHDH was identified and engineered for enantioselective biocatalytic thionation of various aryl‐ and alkyl‐substituted epoxides on a preparative scale, affording the corresponding thiiranes in up to 43 % isolated yield and 98 % ee. Large‐scale synthesis and useful transformations of chiral thiiranes were also performed to demonstrate the utility and scalability of the biocatalytic thionation strategy. [ABSTRACT FROM AUTHOR]

The generation of cross-linked enzyme crystals is a very attractive method for immobilization of enzymes displaying high crystalizability. However, the commonly used cross-linker glutaraldehyde is not always compatible with enzyme activity. Therefore, we previously reported the engineering of halohydrin dehalogenase HheG from Ilumatobacter coccineus to enable thiol-specific cross-linking during CLEC generation by insertion of cysteine residues in the crystal contact. To broaden the applicability of this approach, herein crystal contact engineering of HheG has been performed to incorporate additional lysine residues as defined cross-linking sites for CLEC generation. Using the primary amine-specific cross-linker dithiobis(succinimidyl propionate) (DSP), CLECs of HheG variant V46K were obtained that displayed a high gain in thermal stability compared to wild-type HheG, while using only a low cross-linker concentration. Moreover, respective V46K CLECs exhibited a 10 K higher reaction temperature optimum as well as significantly improved activity and stability at acidic pH and in the presence of organic co-solvents. Overall, our study demonstrates that lysine-specific cross-linkers can also be used as an alternative to glutaraldehyde for stable CLEC generation of halohydrin dehalogenases, and that cross-linking efficiency is significantly improved upon crystal contact engineering. [ABSTRACT FROM AUTHOR]

Although the application of organic solvents in biocatalysis is well explored, in‐depth understanding of the interactions of solvent with proteins, in particular oligomeric ones, is still scant. Understanding these interactions is essential in tailoring enzymes for industrially relevant catalysis in nonaqueous media. In our study, the homotetrameric enzyme halohydrin dehalogenase (HHDH) from Agrobacterium radiobacter AD1 (HheC) was investigated, as a model system, in DMSO/water solvent mixtures. DMSO, the most commonly used co‐solvent for biocatalytic transformations, was found to act as a mixed‐type inhibitor with a prevalent competitive contribution. Even 5 % (v/v) DMSO inhibits the activity of HheC by half. Molecular dynamics (MD) simulations showed that DMSO keeps close to Ser‐Tyr catalytic residues forming alternate H‐bonds with them. Stability measurements paired with differential scanning calorimetry, dynamic light scattering methods and MD studies revealed that HheC maintains its structural integrity with as much as 30 % (v/v) DMSO. [ABSTRACT FROM AUTHOR]

We report the discovery of an unusual halohydrin dehalogenase, HHDHamb, that can work under relatively low acidic conditions and extremely low temperatures for the bio‐nitration of epoxides using nitrite as a nitrating agent. The bio‐nitration strategy exhibits high chemo‐, regio‐, and enantioselectivity, catalyzing the kinetic resolution of various epoxides to enantiopure β‐nitroalcohols with nitro‐bearing stereocenters in up to 41 % isolated yield and >99 % enantiomeric excess (ee). Additionally, the bio‐nitration method displays a high reaction efficiency and can be performed on a gram scale. We also solved the crystal structure of HHDHamb to understand the possible structural determinants of chemoselectivity control in the bio‐nitration reaction. [ABSTRACT FROM AUTHOR]

Halohydrin dehalogenases (HHDHs) possess an unnatural activity of introducing functionalities such as N3, CN, NO2 etc. into a molecule through the ring‐opening reaction of epoxides. The enantioselectivity of HHDHs is substrate‐dependent and not always high enough for synthetic applications. B‐group of HHDHs has been neglected in the past, due to observed low enantioselectivity based on performance on a relatively limited number of substrates. Extensive screening of substrates on HheB2 from Mycobacterium sp. GP1 and HheB from Corynebacterium sp. N‐1074 was performed. Several highly enantioselective reactions were discovered (E>200), with HheB showing higher enantioselectivity and activity toward larger panel of substrates compared to HheB2. Enzymes HheB and HheB2 are highly homologous; they differ by only 4 residues. By using site‐directed mutagenesis, residues 120 and 125 were found to be responsible for higher enantioselectivity of HheB compared to HheB2. Computational analysis supported experiments and provided evidence that kinetic and thermodynamic parameters of reactions within HheB enzymes are crucial in determining the observed enantioselectivities. Due to remarkable activity and enantioselectivity, B‐group HHDHs emerged as a catalyst of choice for the synthesis of bulky tertiary alcohols, as shown in this work. [ABSTRACT FROM AUTHOR]

Although biocatalysis has garnered widespread attention in both industrial and academic realms, the enzymatic synthesis of chiral oxetanes remains an underdeveloped field. Halohydrin dehalogenases (HHDHs) are industrially relevant enzymes that have been engineered to accomplish the reversible transformation of epoxides. In our work, a biocatalytic platform was constructed for the stereoselective kinetic resolution of chiral oxetanes and formation of 1,3-disubstituted alcohols. HheC from Agrobacterium radiobacter AD1 was engineered to identify key variants capable of catalyzing the dehalogenation of γ-haloalcohols (via HheC M1-M3) and ring opening of oxetanes (via HheC M4-M5) to access both (R)- and (S)-configured products with high stereoselectivity and remarkable catalytic activity, yielding up to 49% with enantioselectivities exceeding 99% ee and E>200. The current strategy is broadly applicable as demonstrated by expansion of substrate scope to include up to 18 examples for dehalogenations and 16 examples for ring opening. Additionally, the functionalized products are versatile building blocks for pharmaceutical applications. To shed light on the molecular recognition mechanisms for the relevant variants, molecular dynamic (MD) simulations were performed. The current strategy expands the scope of HHDH-catalyzed chiral oxetane ring constructions, offering efficient access to both enantiomers of chiral oxetanes and 1,3-disubstituted alcohols., (© 2024 Wiley‐VCH GmbH.)

A major drawback for practical application of halohydrin dehalogenase HheG in biocatalysis is its rather low thermal stability and low organic solvent tolerance. We therefore pursued a stabilization of HheG via immobilization as cross‐linked enzyme crystals. Since glutaraldehyde inactivates HheG, we introduced a cysteine residue in the crystal interface, which enabled thiol‐specific cross‐linking at well‐defined cross‐linking sites. Variant HheG D114C displayed improved crystallizability and yielded stable and catalytically active CLECs using bis‐maleimidoethane as cross‐linker. Effective cross‐linking at the predefined site could be confirmed via the CLEC crystal structure. Compared to soluble enzyme, the CLECs displayed significantly improved stability and activity at higher temperatures, lower pH values and in the presence of water‐miscible organic solvents, which enabled their reuse over 21 days in the azidolysis of cyclohexene oxide. [ABSTRACT FROM AUTHOR]

The catalytic reaction of enzyme is carried out inside the cell and the enzyme activity is detected outside the cell using fluorescence spectroscopy. [Display omitted] • Dehalogenase has value in environmental protection and industrial syntheses. • The deep sea is rich in dehalogenase resources. • Halohydrin dehalogenase can catalyze different substrates to produce or consume protons. • Halohydrin dehalogenase activity can be detected using fluorescence assay. • The adaptability to marine environments of the method was studied. Halohydrin dehalogenase (HHDH) can degrade 1,2,3-trichloropropanol and other halogenated alcohols. These enzymes exhibit stereoselectivity and can be used to produce pure chiral epoxides and β-substituted alcohols of industrial value. The discovery of novel HHDHs from microorganisms in niche environments including the sea requires in-situ analysis of HHDH activity. Owing to its high practical value, HheC was selected as a target enzyme to establish an intact-cell method for detecting HHDH activity based on fluorescence spectroscopy. This approach relied on the principle that HHDH activity can change the pH in a system with weak buffering capacity, and that the fluorescence intensity of certain dyes is influenced by pH. The marine adaptability of the assay was subjected to preliminary investigation. The method is simple, rapid, and highly sensitive, as verified using gas chromatography-mass spectrometry. The detection limit of the method was OD 600 (optical density at a 600 nm) of 0.025 for microorganisms containing HHDH which have activity comparable to that of HheC. With some improvements, this method holds potential for in-situ analysis and mining of marine microorganisms containing HHDH. [ABSTRACT FROM AUTHOR]

The generation of cross-linked enzyme crystals is a very attractive method for immobilization of enzymes displaying high crystalizability. However, the commonly used cross-linker glutaraldehyde is not always compatible with enzyme activity. Therefore, we previously reported the engineering of halohydrin dehalogenase HheG from Ilumatobacter coccineus to enable thiol-specific cross-linking during CLEC generation by insertion of cysteine residues in the crystal contact. To broaden the applicability of this approach, herein crystal contact engineering of HheG has been performed to incorporate additional lysine residues as defined cross-linking sites for CLEC generation. Using the primary amine-specific cross-linker dithiobis(succinimidyl propionate) (DSP), CLECs of HheG variant V46K were obtained that displayed a high gain in thermal stability compared to wild-type HheG, while using only a low cross-linker concentration. Moreover, respective V46K CLECs exhibited a 10 K higher reaction temperature optimum as well as significantly improved activity and stability at acidic pH and in the presence of organic co-solvents. Overall, our study demonstrates that lysine-specific cross-linkers can also be used as an alternative to glutaraldehyde for stable CLEC generation of halohydrin dehalogenases, and that cross-linking efficiency is significantly improved upon crystal contact engineering.

Halohydrin dehalogenases (HHDHs) are promising enzymes for application in biocatalysis due to their promiscuous epoxide ring‐opening activity with various anionic nucleophiles. So far, seven different HHDH subtypes A to G have been reported with subtype D containing the by far largest number of enzymes. Moreover, several characterized members of subtype D have been reported to display outstanding characteristics such as high catalytic activity, broad substrate spectra or remarkable thermal stability. Yet, no structure of a D‐type HHDH has been reported to date that could be used to investigate and understand those features on a molecular level. We therefore solved the crystal structure of HheD2 from gamma proteobacterium HTCC2207 at 1.6 Å resolution and used it as a starting point for targeted mutagenesis in combination with molecular dynamics (MD) simulation, in order to study the low thermal stability of HheD2 in comparison with other members of subtype D. This revealed a hydrogen bond between conserved residues Q160 and D198 to be connected with a high catalytic activity of this enzyme. Moreover, a flexible surface region containing two α‐helices was identified to impact thermal stability of HheD2. Exchange of this surface region by residues of HheD3 yielded a variant with 10 °C higher melting temperature and reaction temperature optimum. Overall, our results provide important insights into the structure–function relationship of HheD2 and presumably for other D‐type HHDHs. Databases: Structural data are available in PDB database under the accession number 7B73. [ABSTRACT FROM AUTHOR]

Kinetic resolution of a series of fluorinated styrene oxide derivatives was studied using halohydrin dehalogenase. A mutant HheC-W249P catalysed nucleophilic ring-opening with azide and cyanide ions with excellent enantioselectivity (E-values up to >200), which gives access to various enantiopure β-substituted alcohols and epoxides. It was found that the enzyme tolerates substrates in concentrations over 50 mM. However, different side reactions were observed at elevated concentrations and with prolonged reaction time. The biocatalytic azidolysis and cyanolysis of racemic 4-trifluoromethylstyrene oxide were performed on preparative scale, affording (R)-2-azido-1-(4-trifluoromethyl-phenyl)-ethanol in 38% yield and 97% ee, and (S)-3-hydroxy-3-(4-trifluoromethyl-phenyl)-propionitrile in 30% yield and 98% ee.

Halohydrin dehalogenases (HHDHs) are valuable biocatalysts for the synthesis of enantiopure benzyl glycidyl ether (BGE) and its derivatives, which are important synthetic intermediates for anti-cancer and anti-obesity drugs. However, all the reported HHDHs exhibit low enantioselectivity. In this study, we screened site-saturation mutagenesis libraries of Ab HHDH at positions R89, A136, V137, P178, N179, F180, I181, Y186 and F187 for mutants with enhanced enantioselectivity toward BGE. The four improved variant R89V, R89Y, R89K and V137I were identified, and the double mutant R89Y/V137I showed 2.9-fold higher enantioselectivity than the wild type. The regions of HHDH containing the identified mutations were analyzed by homology modeling to explain the changes of enantioselectivity. Kinetic resolution of 20 to 100 mM BGE using whole cells of Escherichia coli expressing the mutant R89Y/V137I resulted in (R)-BGE yields of 42 to 32.5%, with ee >99%. This study improves our understanding of the enantioselectivity of HHDHs and contributes improved biocatalysts for the kinetic resolution of BGE. [ABSTRACT FROM AUTHOR]

Epoxides are widely used chemicals, the determination of which is of paramount importance. Herein, we present an enzyme‐based approach for noninstrumental detection of epoxides in standard solution and environmental samples. Halohydrin dehalogenase (HheC) as a biological recognition element and epichlorohydrin as a model analyte were evaluated for sensing. The detection is based on the color change of the pH indicator dye bromothymol blue caused by the HheC‐catalyzed ring‐opening of the epoxide substrate. The color change is then exploited for the determination of epoxide using a smartphone as an image acquisition and data processing device, eliminating the need for computer‐based image analysis software. The color parameters were systematically evaluated to determine the optimum quantitative analytical parameter. Under optimal conditions, the proposed enzyme‐based detection system showed a linear range of 0.13–2 mM with a detection limit of 0.07 mM and an assay time of 8 Min. In addition, the repeatability expressed as relative standard deviation was found to be below 5% (n = 6). Validation with gas chromatographic analyses showed that the proposed enzyme‐based epoxide detection could be an alternative way in the quantitative determination of epoxides, and particularly useful in resource‐limited settings. [ABSTRACT FROM AUTHOR]

Directed evolution has become an important method to unleash the latent potential of enzymes to make them uniquely suited for human purposes. However, the need for a large reagent volume and sophisticated instrumentation hampers its broad implementation. In an attempt to address this problem, here we report a paper-based high-throughput screening approach that should find broad application in generating desired enzymes. As an example case, the dehalogenation reaction of the halohydrin dehalogenase was adopted for assay development. In addition to visual detection, quantitative measurements were performed by measuring the color intensity of an image that was photographed by a smartphone and processed using ImageJ free software. The proposed method was first validated using a gold standard method and then applied to mutagenesis library screening with reduced consumption of reagents (i.e., ≤ 10 μl per assay) and a shorter assay time. We identified two active mutants (P135A and G137A) with improved activities toward four tested substrates. The assay not only consumes less reagents but also eliminates the need for expensive instrumentation. The proposed method demonstrates the potential of paper-based whole-cell screening coupled with digital image colorimetry as a promising approach for the discovery of industrially important enzymes. Key Points • A frugal method was developed for directed enzyme evolution. • Mutagenesis libraries were successfully screened on a paper platform. • Smartphone imaging was efficiently used to measure enzyme activities. [ABSTRACT FROM AUTHOR]

An efficient biocatalytic approach for enantio‐ and regioselective ring‐opening of styrene oxides with cyanate was developed by using the halohydrin dehalogenase HheC from Agrobacterium radiobacter AD1, generating the corresponding chiral 5‐aryl‐2‐oxazolidinones in up to 47% yield and 90% ee. Additionally, the origin of enantioselectivity and regioselectivity of the HheC‐catalyzed cyanate‐mediated ring‐opening process was uncovered by single enantiomer bioconversions and molecular docking study. [ABSTRACT FROM AUTHOR]

A gene encoding a halohydrin dehalogenase from Pseudomonas pohangensis (Pp HHDH) 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 E R = 9.8 to E S = 21.6) toward PGE. The wild-type Pp HHDH 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 Pp HHDH 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 Pp HHDH. 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 Pp HHDH. [ABSTRACT FROM AUTHOR]

Halohydrin dehalogenases (HHDHs) have attracted much attention due to their ability to synthesize enantiomerically enriched epoxides and β-haloalcohols. However, most of the HHDHs exhibit low enantioselectivity. Here, a HHDH from the alphaproteobacteria isolate 46_93_T64 (AbHHDH), which shows only poor enantioselectivity in the catalytic resolution of rac-PGE (E = 9.9), has been subjected to protein engineering to enhance its enantioselectivity. Eight mutants (R89K, R89Y, V137I, P178A, N179Q, N179L, F187L, F187A) showed better enantioselectivity than the wild type. The best single mutant N179L (E = 93.0) showed a remarkable 9.4-fold increase in the enantioselectivity. Then, the single mutations were combined to produce the double, triple, quadruple, and quintuple mutants. Among the combinational mutants, the best variant (R89Y/N179L) showed an increased E value of up to 48. The E values of the variants N179L and R89Y/N179L for other epoxides 2–7 were 12.2 to > 200, which showed great improvement compared to 1.2 to 10.5 for the wild type. Using the variant N179L, enantiopure (R)-PGE with > 99% ee could be readily prepared, affording a high yield and a high concentration. [ABSTRACT FROM AUTHOR]

Multi-step cascade reactions have gained increasing attention in the biocatalysis field in recent years. In particular, multi-enzymatic cascades can achieve high molecular complexity without workup of reaction intermediates thanks to the enzymes’ intrinsic selectivity; and where enzymes fall short, organo- or metal catalysts can further expand the range of possible synthetic routes. Here, we present two enantiocomplementary (chemo)-enzymatic cascades composed of either a styrene monooxygenase (StyAB) or the Shi epoxidation catalyst for enantioselective alkene epoxidation in the first step, coupled with a halohydrin dehalogenase (HHDH)-catalysed regioselective epoxide ring opening in the second step for the synthesis of chiral aliphatic non-terminal azidoalcohols. Through the controlled formation of two new stereocenters, corresponding azidoalcohol products could be obtained with high regioselectivity and excellent enantioselectivity (99% ee) in the StyAB-HHDH cascade, while product enantiomeric excesses in the Shi-HHDH cascade ranged between 56 and 61%.

A biocatalytic approach towards a range of 4‐aryloxazolidinones is developed using a halohydrin dehalogenase from Ilumatobacter coccineus (HheG) as biocatalyst. The method is based on the HheG‐catalyzed α‐position regioselective ring‐opening of styrene oxide derivatives with cyanate as a nucleophile, producing the corresponding 4‐aryloxazolidinones in moderate to good yields. Synthesis of enantiopure 4‐aryloxazolidinones is also achievable using chiral epoxide materials. [ABSTRACT FROM AUTHOR]

Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) is a key enzyme in preparing β-substituted alcohols and optically pure epoxides. However, the weak thermostability of HheC greatly limits its industrial application and needs to be improved. In this study, a combinatorial smart library was constructed and screened for enhancing the thermostability of HheC. Mutation sites Lys203 and Lys204 located at the protein surface were selected and simultaneously randomized with charged amino acids (Arg, Asp, Glu, Lys), resulting in a combinatorial smart library. After screening a total of only 48 clones, three positive variants CSL1 (Lys203Arg), CSL2 (Lys204Arg) and CSL3 (Lys203Arg/Lys204Arg) were obtained. Compared to the wild type HheC, variant CSL2 exhibited an increase in thermal stability with a nearly 6.9-fold improvement of half-life (τ 1/2, 55°C) at 55 °C without compromising catalytic activity. In addition, the double mutant CSL3 displayed approximately 3.4-fold higher τ 1/2, 55°C value and 1.8-fold higher k cat value toward 1,3-dicholoro-2-propanol (1,3-DCP) than that of the wild-type HheC. Homology modeling analysis highlights that the newly gained hydrogen bonds and optimizing charge-charge interactions at the protein surface might contribute to the overall conformational stabilization, leading to improving the thermostability of HheC. • A combinatorial smart library was constructed for improving thermostability of HheC. • After screening a total of only 48 clones, three positive variants were obtained. • CSL2 and CSL3 show 6.9- and 3.4-fold higher half-life values than that of HheC. • CSL1 and CSL3 exhibit 1.6- and 1.8-fold higher k cat values as compare to HheC. [ABSTRACT FROM AUTHOR]

Halohydrin dehalogenases (HHDHs) are valuable biocatalysts for the synthesis of β‐substituted alcohols based on their epoxide ring‐opening activity with a number of small anionic nucleophiles. In an attempt to further broaden the scope of substrates accepted by these enzymes, a panel of 22 HHDHs was investigated in the conversion of aliphatic and aromatic vicinally di‐substituted trans‐epoxides using azide as nucleophile. The majority of these HHDHs was able to convert aliphatic methyl‐substituted epoxide substrates to the corresponding azidoalcohols, in some cases even with absolute regioselectivity. HheG from Ilumatobacter coccineus exhibited also high activity towards sterically more demanding di‐substituted epoxides. This further expands the range of β‐substituted alcohols that are accessible by HHDH catalysis. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Key amino acid residues located at halide ion channels of HheC PS were modified. • The kinetic parameters of HheC PS were regulated. • Optically pure (S)-ECH was biosynthesized for the first time by HheC PS 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) (HheC PS) were modified to regulate the kinetic parameters. HheC PS I81W, F86N and V94R were constructed with the corresponding halide ion channels destroyed. The catalytically efficiencies (k cat / K m) 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. [ABSTRACT FROM AUTHOR]

A gene encoding halohydrin dehalogenase from an alphaproteobacterium (AbHHDH) was identified, cloned and over-expressed in Escherichia coli. AbHHDH was able to catalyze the stereoselective dehalogenation of prochiral and racemic halohydrins. It showed the highest enantioselectivity in the dehalogenation of 20 mM (R,S)-2-bromo-1-phenylethanol, which yielded (S)-2-bromo-1-phenylethanol with 99% ee and 34.5% yield. Moreover, AbHHDH catalyzed the azidolysis of epoxides with low to moderate (S)-enantioselectivity. The highest enantioselectivity (E = 18.6) was observed when (R,S)-benzyl glycidyl ether was used as the substrate. A sequential kinetic resolution catalyzed by HHDH was employed for the synthesis of chiral 1-chloro-3-phenoxy-2-propanol. We prepared enantiopure (S)-isomer with a high enantiopurity of ee > 99% and a yield of 30.7% (E-value: 21.3) by kinetic resolution of 20 mM substrate. The (S)-isomer with 99% ee readily obtained from 40 to 150 mM (R,S)-1-chloro-3-phenoxy-2-propanol. Taken together, the results of this study demonstrate the applicability of this HHDH for the production of optically active compounds. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Cloning and expression of a novel halohydrin dehalogenase (HHDH) from P. umsongensis. • HHDH-Pu exhibits high activity towards the epoxides. • HHDH-Pu catalyzed azidolysis of epoxides with low to moderate R -enantioselectivity. • (S)-PGE could be obtained with >99% ee and 23.5% yield from 40 mM rac -PGE. 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−, N 3 −, NO 2 −, Br− or SCN− were accepted. N 3 − was selected as the optimal nucleophile. Some parameters that may alter this bio-resolution, such as the concentration of N 3 −, 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 [ABSTRACT FROM AUTHOR]

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 HheC CLEAs 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, HheC CLEAs maintained more than 70% activity after 10 reusability cycles. The utility of HheC CLEAs 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 HheC CLEAs overcame some disadvantages of free enzymes to become biocatalysts. Together with further engineering of the enzyme, HheC CLEAs could become a promising biocatalyst for the synthesis of valuable chiral compounds. [ABSTRACT FROM AUTHOR]

Abstract: 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.Graphical Abstract: [ABSTRACT FROM AUTHOR]

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 ) inactivation at 65 °C, 18 °C increase in apparent T 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. [ABSTRACT FROM AUTHOR]

The immobilization conditions of halohydrin dehalogenase (HHDH) by the sodium alginate were studied. The optimum conditions of immobilization process were as follows: sodium alginate concentration 3.5%, HHDH enzymatic amount 2.4 x 104 U/mL, glutaraldehyde concentration 1.00%, calcium chloride solution concentration 1.5% and immobilization time 2 h. Under the optimum immobilization conditions, the encapsulation efficiency of HHDH was 52.1%, the activity recovery of HHDH was 75.2%. After five batches of intermittent catalytic synthesis of (R)-4-cyano-3-hydroxybutyric acid ethyl (ATS-5), the residual activity of immobilized HHDH was 51.2%. After the continuous catalytic synthesis of ATS-5 for 10 d, the substrate conversion efficiency was 90.6%, the distillation purification yield of ATS-5 was 98.2%, the purity and optical purity of ATS-5 were 99.3% and 99.1%, respectively. No obvious activity loss was obtained for immobilized HHDH after 80 d storage at 4 °C. [ABSTRACT FROM AUTHOR]