Increasing the Reaction Rate of Hydroxynitrile Lyase from Hevea brasiliensis toward Mandelonitrile by Copying Active Site Residues from an Esterase that Accepts Aromatic Esters

The natural substrate of hydroxynitrile lyase from rubber tree (HbHNL, Hevea brasiliensis) is acetone cyanohydrin, but synthetic applications usually involve aromatic cyanohydrins such as mandelonitrile. To increase the activity of HbHNL toward this unnatural substrate, we replaced active site residues in HbHNL with the corresponding ones from esterase SABP2 (salicylic acid binding protein 2). Although this enzyme catalyzes a different reaction (hydrolysis of esters), its natural substrate (methyl salicylate) contains an aromatic ring. Three of the eleven single‐amino‐acid‐substitution variants of HbHNL reacted more rapidly with mandelonitrile. The best was HbHNL‐L121Y, with a k_cat 4.2 times higher and high enantioselectivity. Site‐saturation mutagenesis at position 121 identified three other improved variants. We hypothesize that the smaller active site orients the aromatic substrate more productively.     

Hydroxynitrile Lyases with α/β-Hydrolase Fold: Two Enzymes with Almost Identical 3D Structures but Opposite Enantioselectivities and Different Reaction Mechanisms

Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins to yield hydrocyanic acid (HCN) and the respective carbonyl compound and are key enzymes in the process of cyanogenesis in plants. In organic syntheses, HNLs are used as biocatalysts for the formation of enantiopure cyanohydrins. We determined the structure of the recently identified, R‐selective HNL from Arabidopsis thaliana (AtHNL) at a crystallographic resolution of 2.5 Å. The structure exhibits an α/β‐hydrolase fold, very similar to the homologous, but S‐selective, HNL from Hevea brasiliensis (HbHNL). The similarities also extend to the active sites of these enzymes, with a Ser‐His‐Asp catalytic triad present in all three cases. In order to elucidate the mode of substrate binding and to understand the unexpected opposite enantioselectivity of AtHNL, complexes of the enzyme with both (R)‐ and (S)‐mandelonitrile were modeled using molecular docking simulations. Compared to the complex of HbHNL with (S)‐mandelonitrile, the calculations produced an approximate mirror image binding mode of the substrate with the phenyl rings located at very similar positions, but with the cyano groups pointing in opposite directions. A catalytic mechanism for AtHNL is proposed, in which His236 from the catalytic triad acts as a general base and the emerging negative charge on the cyano group is stabilized by main‐chain amide groups and an α‐helix dipole very similar to α/β‐hydrolases. This mechanistic proposal is additionally supported by mutagenesis studies.     

Enantioselective Cascade Biocatalysis for Deracemization of Racemic β-Amino Alcohols to Enantiopure (S)-β-Amino Alcohols by Employing Cyclohexylamine Oxidase and ω-Transaminase

Optically active β-amino alcohols are very useful chiral intermediates frequently used in the preparation of pharmaceutically active substances. Here, a novel cyclohexylamine oxidase (ArCHAO) was identified from the genome sequence of Arthrobacter sp. TYUT010-15 with the R-stereoselective deamination activity of β-amino alcohol. ArCHAO was cloned and successfully expressed in E. coli BL21, purified and characterized. Substrate-specific analysis revealed that ArCHAO has high activity (4.15 to 6.34 U mg⁻¹ protein) and excellent enantioselectivity toward the tested β-amino alcohols. By using purified ArCHAO, a wide range of racemic β-amino alcohols were resolved, (S)-β-amino alcohols were obtained in >99 % ee. Deracemization of racemic β-amino alcohols was conducted by ArCHAO-catalyzed enantioselective deamination and transaminase-catalyzed enantioselective amination to afford (S)-β-amino alcohols in excellent conversion (78–94 %) and enantiomeric excess (>99 %). Preparative-scale deracemization was carried out with 50 mM (6.859 g L⁻¹) racemic 2-amino-2-phenylethanol, (S)-2-amino-2-phenylethanol was obtained in 75 % isolated yield and >99 % ee.     

Yeast‐mediated enantioselective synthesis of chiral R‐(+)‐ and S‐(−)‐1‐phenyl‐1‐butanol from prochiral phenyl n‐propyl ketone in hexane–water biphasic culture

BACKGROUND: The hydrophobic phenyl n‐propyl ketone was used as a model compound to examine alcohol dehydrogenase activity in Saccharomyces cerevisiae mediated cell culture. Parameters such as pH, hexane‐to‐water volume percentage, and the amount of cofactor Zn²⁺ ion for either cell growth or reduction were studied to see their effect on the enantioselectivity toward the product R‐(+)‐ or S‐(?)‐1‐phenyl‐1‐butanol. RESULTS: The pH for cell growth in aqueous culture was 7.0, while the pH for reduction in the aqueous portion of the biphasic culture was 5.0. Without Zn²⁺ ion the biphasic cultures of middle to high hexane‐to‐water volume percentage exhibited an R‐(+)‐1‐phenyl‐1‐butanol enantiomeric excess of 53.7% to > 99%. Without Zn²⁺ ion the biphasic cultures at low hexane‐to‐water volume percentage possessed an S‐(?)‐1‐phenyl‐1‐butanol enantiomeric excess of 14.5–46.5%. Exclusively, the enantioselectivity for biphasic cultures containing Zn²⁺ ion was an S‐(?)‐1‐phenyl‐1‐butanol enantiomeric excess of 27.5% to > 99%. Reduction mediated in aqueous culture with varied amount of Zn²⁺ ion by the yeast Candida utilis also showed an S‐(?)‐1‐phenyl‐1‐butanol enantiomeric excess of 79.2–95.4%. CONCLUSION: The enantioselectivity of S. cerevisiae mediated biphasic culture reduction of phenyl n‐propyl ketone can be manipulated through the cofactor Zn²⁺ ion and the hexane volume percentage of the biphasic culture. Copyright © 2008 Society of Chemical Industry     

Resolution of N‐hydroxymethyl vince lactam catalyzed by lipase in organic solvent

BACKGROUND: The enantiomers of N‐hydroxymethyl vince lactam are important intermediates during the synthesis of chiral drugs. The preparation of its single enantiomer can be performed through enzymatic resolution. The aim of this work is to obtain (1S, 4R)‐N‐hydroxymethyl vince lactam with high enantiomeric purity via lipase‐catalyzed enantioselective transesterification in organic solvents. To achieve this, effects of various reaction conditions (including lipase sources, acyl donor, substrate molar ratio, organic solvent, temperature, and water activity) on the enzyme activity as well as enantioselectivity were investigated. RESULTS: The results of the study showed that the enantiopreference for all the selected enzymes was (4S, 1R)‐N‐hydroxymethyl vince lactam in enantioselective transesterification of racemic N‐hydroxymethyl vince lactam. Under the selected optimum conditions, the highest enantioselectivity (E = 33.8) was obtained with a higher enzyme activity (20.3 µmol g^?1 min^?1) for Mucor miehei lipase (MML) when vinyl valerate was used as the acyl donor. Besides, the remained (1S, 4R)‐N‐hydroxymethyl vince lactam with high enantiomeric purity (ee > 99%) was obtained when the conversion was about 60%. CONCLUSION: The results obtained clearly demonstrated potential for industrial application of lipase in resolution of N‐hydroxymethyl vince lactam through enantioselective transesterification. © 2012 Society of Chemical Industry     

Enhancement of enantioselectivity and reaction rate on the synthesis of (S)‐ketoprofen hydroxyalkyl ester in organic solvents via isopropanol‐dried immobilized lipase

Lipase‐catalyzed enantioselective esterification between (R,S)‐ketoprofen and alkanediol in organic solvents was developed to produce (S)‐ketoprofen hydroxyalkyl esters. The acyl acceptor of 1,6‐hexanediol for the resolution of (R,S)‐ketoprofen yielded only the enantioselectivity (the enantiomeric ratio of initial rate for (S)‐ketoprofen to that of (R)‐ketoprofen) V_S/V_R = 8, when crude Lipase MY originating from Candida rugosa was used. However, isopropanol‐dried immobilized lipases (IPA‐dried IM‐lipase) effectively enhanced the enantioselectivity to greater than 20 in the esterification of (R,S)‐ketoprofen when 1,4‐butanediol, 1,5‐pentanediol or 1,6‐hexanediol was employed. IPA‐dried IM‐lipase and isooctane were selected to use for optimally immobilized lipase and reaction medium, respectively. The IPA‐dried IM‐lipase exhibited the highest enantioselectivity, E = 26.7, to the (S)‐enantiomer with 1,5‐pentanediol and the best enzyme activity to the (S)‐enantiomer with 1,4‐butanediol. The finding indicates that the carbon chain length of the alkanediol strongly affected the enzyme activity and enantioselectivity of lipase‐catalyzed esterification. A maximum enantioselectivity of 37 at 27 °C was generated by IPA‐dried IM‐lipase for the enantioselective esterification of racemic ketoprofen with 1,4‐butanediol. IPA‐dried IM‐lipase can effectively increase the enantioselectivity of lipase. Copyright © 2005 Society of Chemical Industry     

Effect of Glycosylation on the Biocatalytic Properties of Hydroxynitrile Lyase from the Passion Fruit,Passiflora edulis: A Comparison of Natural and Recombinant Enzymes

A hydroxynitrile lyase from the passion fruit Passiflora edulis (PeHNL) was isolated from the leaves and showed high stability in biphasic co‐organic solvent systems for cyanohydrin synthesis. Cyanohydrins are important building blocks for the production of fine chemicals and pharmaceuticals. Thus, to enhance production yields of PeHNL for industrial applications, we cloned and expressed recombinant PeHNL in Escherichia coli BL21(DE3) and Pichia pastoris GS115 cells without a signal peptide sequence. The aim of this study is to determine the effect of N‐glycosylation on enzyme stability and catalytic properties in microbial expression systems. PeHNL from leaves (PeHNL‐N) and that expressed in P. pastoris (PeHNL‐P) were glycosylated, whereas that expressed in E. coli (PeHNL‐E) was not. The enzymes PeHNL‐N and PeHNL‐P showed much better thermostability, pH stability, and organic solvent tolerance than the deglycosylated enzyme PeHNL‐E and the deglycosylated mutant N105Q from P. pastoris (PeHNL‐P‐N105Q). The glycosylated PeHNL‐P also efficiently performed transcyanation of (R)‐mandelonitrile with a 98 % enantiomeric excess in a biphasic system with diisopropyl ether. These data demonstrate the efficacy of these methods for improving enzyme expression and stability for industrial application through N‐glycosylation.     

Biochemical Characterization of Purified Esterase from Soybean (Glycine max) Seed

Alkaline esterase (carboxylic‐ester hydrolases; EC 3.1.1.1) extracted from germinated soybean seeds (Glycine max) was purified approximately 3.6 times by chromatography in a DEAE‐cellulose anion exchange column and filtration in Sephadex G100 gel. The molecular mass of the enzyme was estimated at 45 kDa by gel electrophoresis (SDS‐PAGE). The purified enzyme showed a specific activity of 5.6 U mg^?1 using p‐nitrophenyl butyrate as substrate. The esterase showed optimal activity at 47 °C in moderately alkaline pH, low stability in temperatures higher than 50 °C, and high stability at pH values between 6 and 9.5. The Ca²⁺ and Co²⁺ ions proved to have a positive effect on enzyme activity; however, Hg²⁺ completely inhibited esterase activity. Using p‐nitrophenyl butyrate as substrate, the enzyme showed a K_m of 0.39 mM, V_max of 31.5 mM mg^?1 min^?1 and k_cat 7.60 × 10⁶ s^?1. Regarding substrate affinity, the enzyme showed greater activity for substrates containing short‐chain fatty acids, especially p‐nitrophenyl acetate. Such characteristics give the enzyme great potential for application in the production of low molecular weight esters, in the food industry, and in chemical products. This enzyme is another new member of the family of lipases and esterases from vegetable seeds with high activity and stability in alkaline pH.     

Determination of the Enantioselectivity for Kinetically Controlled Condensations Catalysed by Amidases and Peptidases

For kinetically controlled synthetic reactions catalysed by amidases and peptidases, the enantio‐ or stereoselectivity determined from initial rates with racemic mixtures (E_{syn, rac}) was found to differ from the enantioselectivity determined from measurements with isolated enantiomeric nucleophiles (E_syn). This was observed for kinetically controlled condensation of R‐phenyglycineamide with S‐, R‐ and racemic Phe and S‐, R‐ and racemic Leu catalysed by penicillin amidase from E. coli and for kinetically controlled condensation of N^α‐acetyl‐S‐tyrosine ethyl ester with S‐, R‐ and racemic AlaNH₂ catalysed by bovine α‐chymotrypsin. It is shown that only E_{syn, rac} determined with racemic nucleophiles is an intrinsic enzyme property which should be used to study the influence of the primary structure, physicochemical parameters and immobilisation on biocatalyst enantioselectivity in kinetically controlled synthetic reactions catalysed by these enzymes.     

Improvement of enantioselectivity and stability of Klebsiella oxytoca hydrolase immobilized on Eupergit C 250L

BACKGROUND: A simple procedure was employed to covalently immobilize a Klebsiella oxytoca hydrolase (SNSM‐87) onto epoxy‐activated supports of Eupergit C 250L via multipoint covalent attachment. The resultant biocatalyst was explored for the hydrolytic resolution of a variety of (R,S)‐2‐hydroxycarboxylic acid ethyl esters. RESULTS: With the hydrolytic resolution of (R,S)‐ethyl mandelate in biphasic media as the model system, optimal conditions of 55 °C, pH 6 buffer and isooctane as the organic phase were selected for improving the enzyme stability (activity retained from 10% to 50% at 96 h) and enantioselectivity (V_SV_R^?1 value enhanced from 44 to 319) in comparison to the performance of free enzyme. Moreover, the immobilized enzyme retained its activity and enantioselectivity after eight cycles of hydrolysis at 55 °C. When applying the resolution process to other (R,S)‐2‐hydroxycarboxylic acid ethyl esters, 2.4‐ to 4.0‐fold enhancements of the enantioselectivity in general were obtainable. CONCLUSIONS: The enantioselectivity enhancement, good reusability and easy recovery after reaction indicate that the immobilized SNSM‐87 may have the potential as an industrial biocatalyst for the preparation of optically pure 2‐hydroxycarboxylic acids. Copyright © 2008 Society of Chemical Industry     

Profiles of xylose reductase,xylitol dehydrogenase and xylitol production under different oxygen transfer volumetric coefficient values

BACKGROUND: Xylitol is a sugar alcohol (polyalcohol) with many interesting properties for pharmaceutical and food products. It is currently produced by a chemical process, which has some disadvantages such as high energy requirement. Therefore microbiological production of xylitol has been studied as an alternative, but its viability is dependent on optimisation of the fermentation variables. Among these, aeration is fundamental, because xylitol is produced only under adequate oxygen availability. In most experiments with xylitol‐producing yeasts, low oxygen transfer volumetric coefficient (K_La) values are used to maintain microaerobic conditions. However, in the present study the use of relatively high K_La values resulted in high xylitol production. The effect of aeration was also evaluated via the profiles of xylose reductase (XR) and xylitol dehydrogenase (XD) activities during the experiments. RESULTS: The highest XR specific activity (1.45 ± 0.21 U mg_protein^?1) was achieved during the experiment with the lowest K_La value (12 h^?1), while the highest XD specific activity (0.19 ± 0.03 U mg_protein^?1) was observed with a K_La value of 25 h^?1. Xylitol production was enhanced when K_La was increased from 12 to 50 h^?1, which resulted in the best condition observed, corresponding to a xylitol volumetric productivity of 1.50 ± 0.08 g_xylitol L^?1 h^?1 and an efficiency of 71 ± 6.0%. CONCLUSION: The results showed that the enzyme activities during xylitol bioproduction depend greatly on the initial K_La value (oxygen availability). This finding supplies important information for further studies in molecular biology and genetic engineering aimed at improving xylitol bioproduction. Copyright © 2008 Society of Chemical Industry     

面包酵母No.6催化不对称合成(2S,5S)-2,5-已二醇反应特性

Baker’s yeast number 6 was selected by screening. It showed good catalytic activity and enantioselectivity for asymmetric reduction of 2,5-hexanedione to produce (2S,5S)-2,5-hexanediol. Gas chromatography-mass spectrometry (GC-MS) revealed that the intermediate was (S)-5-hydroxyhexane-2-one. Reduction of 2,5-hexanedione proceeded in a two-step reaction. The hydroxyketone was initially formed, and this intermediate was further re-duced to the diol. Factors influencing the product yield and the enantiomeric excess of the reduction of 2,5-hexandione catalyzed by baker’s yeast number 6 were investigated. Higher concentration (≤100 mmol•L－1) of 2,5-hexandione did not influence 5-hydroxyhexane-2-one production, but 2,5-hexanediol production was inhibited by excess accumulation (＞30 mmol•L－1) of intermediate. The optimal conditions were glucose as the co-substrate at an initial glucose concentration of 20 g•L－1, 34C, pH 7.0 and cell concentration 60 g•L－1 (cell dry mass). Under the optimal condition and an initial substrate concentration of 30 mmol•L－1, the yield of 2,5-hexandiol was 78.7% and the enantiomeric excess of (2S,5S)-2,5-hexandiol was 94.4% for 24-h reduction.     

Characterization of the Rv3377c Gene Product,a Type‐B Diterpene Cyclase,from the Mycobacterium tuberculosis H37 Genome

The Rv3377c gene from the Mycobacterium tuberculosis H37 genome is specifically limited to those Mycobacterium species that cause tuberculosis. We have demonstrated that the gene product of Rv3377c is a diterpene cyclase that catalyzes the formation of tuberculosinol from geranylgeranyl diphosphate (GGPP). However, the characteristics of this enzyme had not previously been studied in detail with homogeneously purified enzyme. The purified enzyme catalyzed the synthesis of tuberculosinyl diphosphate from GGPP, but it did not bring about the synthesis of tuberculosinol. Optimal conditions for the highest activity were found to be as follows: pH 7.5, 30 °C, Mg^II (0.1 mM ), and Triton X‐100 (0.1 %). Under these conditions, the kinetic values of K_M and k_cat were determined to be 11.7±1.9 μM for GGPP and 12.7±0.7 min^?1, respectively, whereas the specific activity was 186 nmol min^?1 mg^?1. The enzyme activity was inhibited at substrate concentrations higher than 50 μM . The catalytic activity was strongly inhibited by 15‐aza‐dihydrogeranylgeraniol and 5‐isopropyl‐N,N,N,2‐tetramethyl‐4‐(piperidine‐1‐carbonyloxy)benzenaminium chloride (Amo‐1618). The DXDTT_293–297 motif, corresponding to the DXDDTA motif conserved among terpene cyclases, was mutated in order to investigate its function. The middle D295 was found to be the most crucial entity for the catalysis. D293 and two threonine residues function synergistically to enhance the acidity of D295, possibly through hydrogen‐bonding networks. The Rv3377c enzyme could also react with (14R/S)‐14,15‐oxidoGGPP to generate 3α‐ and 3β‐hydroxytuberculosinyl diphosphate. Conformational analyses were carried out with deuterium‐labeled GGPP and oxidoGGPP. We found that GGPP and (14R)‐oxidoGGPP adopted a chair/chair conformation, but (14S)‐oxidoGGPP adopted a boat/chair conformation. Interestingly, the conformations of oxidoGGPP for the A‐ring formation are the opposite of those of oxidosqualene when it is used as a substrate by squalene cyclases for the biosynthesis of hopene and tetrahymanol. (3R)‐Oxidosqualene is folded in a boat conformation, whereas (3S)‐2,3‐oxidosqualene folds into a chair conformation, for the formation of the A‐rings of the hopene and tetrahymanol skeletons, respectively.     

Beet molasses based exponential feeding strategy for thermostable glucose isomerase production by recombinant Escherichia coli BL21 (DE3)

BACKGROUND: The effects of pretreated beet molasses based feeding strategies on thermostable glucose isomerase (GI) production by recombinant Escherichia coli BL21 (DE3) pLysS were investigated. RESULTS: The thermostable GI encoding gene of Thermus thermophilus (xyl_A) was recombined with pRSETA vector, and the pRSETA::xyl_A obtained was transferred into E.coli BL21 (DE3) pLysS and used for GI production. The highest soluble GI activity was obtained at t = 30 h, as A = 16 400 U L^?1 (20.6 U mg^?1 protein) under molasses based fed‐batch operation, with a specific growth rate µ = 0.1 h^?1 (M‐0.1); on the other hand, the highest cell concentration was obtained at µ = 0.15 h^?1 operation as 9.6 g L^?1 at t = 32 h. The highest oxygen uptake was 4.57 mol m^?3 s^?1 at M‐0.1 operation. CONCLUSIONS: Molasses based fed‐batch operations were more successful in terms of cell concentration and thermostable enzyme production due to the existence of a natural sugar inducer, galactose, in the molasses composition. This study demonstrates the significance of proper feeding strategy development for over‐production of enzymes by recombinant E. coli strains. © 2012 Society of Chemical Industry     

Lipase‐catalyzed enantioselective esterification of S(+)‐naproxen ester prodrugs in cyclohexane

A lipase‐catalyzed enantioselective esterification process in cyclohexane was developed for the synthesis of S(+)‐naproxen ester prodrugs containing the moiety of N,N‐dialkylamino, ethylene glycol or alkyl ether of ethylene glycol. A high enantiomeric ratio of 44 was obtained when di(ethylene glycol) was selected as the best acyl acceptor. A reversible ping‐pong Bi Bi mechanism has been employed to elucidate the enzymatic behavior of the initial conversion rate for S(+)‐naproxen and the time‐course conversions for both enantiomers. Improvement of the enzyme activity was demonstrated when alcohol in excess of its cyclohexane solubility limit was used. The application of excess racemic naproxen in the presence of solid substrate suspensions showed enhanced productivity and enantioselectivity for the desired S(+)‐ester. Studies of the recovery and racemization of the remaining R(−)‐naproxen are also reported. © 1999 Society of Chemical Industry     

Enhancing Effect of Calix[4]arene Amide Derivatives on Lipase Performance in Enantioselective Hydrolysis of Racemic Arylpropionic Acid Methyl Esters

Calix[4]arene amide derivatives were employed as new additives within the sol-gel encapsulation of lipase from Candida rugosa (CRL) to improve its catalytic properties. Evaluation of catalytic activity of the encapsulated lipases was acheived by enantioselective hydrolysis of both racemates, Naproxen methyl ester and 2-phenoxypropionic acid methyl ester, in aqueous buffer solution/isooctane reaction system. Results show that enantioselectivity was improved by using calix[4]arene amide derivatives-based encapsulated lipases. The reaction of naproxen methyl ester resulted in 47.6% conversion (x) in 24 h with 88.9% enantiomeric excess of substrate (ee_s), analogous to an enantioselectivity (E) value of 297 (E = 137 for the encapsulated free enzyme). The conversion of 2-phenoxypropionic acid methyl ester, obtained was 48.4% with E value of 327, enantiomeric excess of substrate (ee_s) of 92% for the reaction time of 1 h (E = 211 for the encapsulated free enzyme).     

Production of (S)-β-Nitro Alcohols by Enantioselective C−C Bond Cleavage with an R-Selective Hydroxynitrile Lyase

Hydroxynitrile lyase (HNL)-catalysed stereoselective synthesis of β-nitro alcohols from aldehydes and nitroalkanes is considered an efficient biocatalytic approach. However, only one S-selective HNL—Hevea brasiliensis (HbHNL)—exists that is appropriate for the synthesis of (S)-β-nitro alcohols from the corresponding aldehydes. Further, synthesis catalysed by HbHNL is limited by low specific activity and moderate yields. We have prepared a number of (S)-β-nitro alcohols, by kinetic resolution with the aid of an R-selective HNL from Arabidopsis thaliana (AtHNL). Optimization of the reaction conditions for AtHNL-catalysed stereoselective C−C bond cleavage of racemic 2-nitro-1-phenylethanol (NPE) produced (S)-NPE (together with benzaldehyde and nitromethane, largely from the R enantiomer) in up to 99 % ee and with 47 % conversion. This is the fastest HNL-catalysed route known so far for the synthesis of a series of (S)-β-nitro alcohols. This approach widens the application of AtHNL for the synthesis not only of (R)- but also of (S)-β-nitro alcohols from the appropriate substrates. Without the need for the discovery of a new enzyme, but rather by use of a retro-Henry approach, it was used to generate a number of (S)-β-nitro alcohols by taking advantage of the substrate selectivity of AtHNL.     

An Esterase with Superior Activity and Enantioselectivity towards 1,2‐O‐Isopropylideneglycerol Esters Obtained by Protein Design

The Escherichia coli esterase YbfF displays high activity towards 1,2‐O‐isopropylideneglycerol (IPG) butyrate and IPG caprylate, and prefers the R‐enantiomer of these substrates, producing the S‐enantiomer of the IPG product in excess. To improve the potential of the enzyme for the kinetic resolution of racemic esters of IPG, an enhancement of the activity and enantioselectivity would be highly desirable. Molecular docking of the R‐enantiomer of both IPG esters into the active site of YbfF allowed the identification of proximal YbfF active site residues. Four residues (25, 124, 185 and 235) were selected as targets for mutagenesis, in order to enhance YbfF activity and enantioselectivity towards IPG esters. Random mutagenesis at positions 25, 124, 185 and 235 yielded several best YbfF variants with enhanced activity and enantioselectivity towards IPG esters. The best YbfF mutant, W235I, exhibited a 2‐fold higher enantioselectivity than wild‐type YbfF, with an E=38 for IPG butyrate and an E=77 for IPG caprylate. Molecular docking experiments further support the enhanced enantioselectivity shown experimentally and the structural effects of this amino acid substitution on the active site of YbfF are provided. The engineered W235I mutant is an attractive catalyst for practical applications in the kinetic resolution of IPG esters.     

Stereoselective hydride transfer by aryl-alcohol oxidase, a member of the GMC superfamily

Primary alcohol oxidation by aryl‐alcohol oxidase (AAO), a flavoenzyme providing H₂O₂ to ligninolytic peroxidases, is produced by concerted proton and hydride transfers, as shown by substrate and solvent kinetic isotope effects (KIEs). Interestingly, when the reaction was investigated with synthesized (R)‐ and (S)‐α‐deuterated p‐methoxybenzyl alcohol, a primary KIE (≈6) was observed only for the R enantiomer, revealing that the hydride transfer is highly stereoselective. Docking of p‐methoxybenzyl alcohol at the buried crystal active site, together with QM/MM calculations, showed that this stereoselectivity is due to the position of the hydride‐ and proton‐receiving atoms (flavin N5 and His502 Nε, respectively) relative to the alcohol Cα‐substituents, and to the concerted nature of transfer (the pro‐S orientation corresponding to a 6 kcal mol^?1 penalty with respect to the pro‐R orientation). The role of His502 is supported by the lower activity (by three orders of magnitude) of the H502A variant. The above stereoselectivity was also observed, although activities were much lower, in AAO reactions with secondary aryl alcohols (over 98 % excess of the R enantiomer after treatment of racemic 1‐(p‐methoxyphenyl)ethanol, as shown by chiral HPLC) and especially with use of the F501A variant. This variant has an enlarged active site that allow better accommodation of the α‐substituents, resulting in higher stereoselectivity (S/R ratios) than is seen with AAO. High enantioselectivity in a member of the GMC oxidoreductase superfamily is reported for the first time, and shows the potential for engineering of AAO for deracemization purposes.     

Manipulating the expression rate and enantioselectivity of an epoxide hydrolase by using directed evolution

We describe here a strategy to improve the expression efficiency and enantioselectivity of Aspergillus niger epoxide hydrolase (ANEH) by directed evolution. Based on a blue‐colony screening system using the LacZα (β‐galactosidase α peptide) complementation solubility reporter, several ANEH variants out of 15 000 transformants from a random‐mutagenesis library were identified that show improved recombinant expression in E. coli. Among them, Pro221Ser was subsequently used as a template for iterative saturation mutagenesis (ISM) at sites around the ANEH binding pocket. Following four rounds of ISM, a highly enantioselective mutant was identified that catalyzes the hydrolytic kinetic resolution of racemic glycidyl phenyl ether with a selectivity factor of E=160 in favor of the (S)‐diol compared to WT ANEH characterized by E=4.6. Expression of this mutant is 50 times higher than that of WT ANEH. It also serves as an excellent stereoselective catalyst in the hydrolytic kinetic resolution and desymmetrization of several other structurally diverse epoxides.