Enzyme Enantioselectivity Research Articles

Solvent effects on the enantioselectivity of the thermophilic lipase QLM in the resolution of ( R, S)-2-octanol and ( R, S)-2-pentanol

The thermophilic lipase QLM-catalyzed resolution of ( R, S)-2-octanol and ( R, S)-2-pentanol via transesterification was carried out in various organic solvents, and the solvent effects on the enzyme's enantioselectivity were investigated. A significant negative correlation between the enantiomeric ratio, E, and the size of the solvent molecules was observed. The highest E value, 21, was obtained in the small molecular-sized solvent dichloromethane when ( R, S)-2-octanol was resolved with vinyl acetate as acyl donor. Thermodynamic analysis indicated that the difference in activation free energy between the two enantiomers was about 25.5% lower in dichloromethane than in the solvent-free system, and the change in the difference in activation entropy between the enantiomers was the main contributor to the changes in E values with the molecular size of solvents.

Characterization and catalytic activity of free and immobilized lipase from Aspergillus niger: a comparative study

Aspergillus niger lipase was immobilized by deposition on Celite and the hydrolytic and esterification activities, stability and enantioselectivity of both free and immobilized enzyme were compared. Both the free and immobilized preparations showed similar biochemical properties, with maximum activity at pH 6.0 and a temperature of 30-40 ºC. The most important effects observed when the lipase was immobilized were thermal stability and an improved esterification activity during the reaction of (R,S)-ibuprofen with 1-propanol in isooctane. Moreover, immobilized Aspergillus niger lipase maintained an esterification activity of at least 73% after 5 days of storage at 40 ºC and can be recycled and reused at least 6 times.

A genetic selection system for evolving enantioselectivity of enzymes

As an alternative to screening in the directed evolution of enantioselective enzymes, a selection system has been implemented for a lipase-catalyzed hydrolytic kinetic resolution of a chiral ester.

Candida rugosa lipase‐catalysed kinetic resolution of 2‐substituted‐aryloxyacetic esters with dimethylsulfoxide and isopropanol as additives

Candida rugosa lipase-catalysed hydrolysis of three different 2-substituted-aryloxyacetic esters was performed in aqueous buffer containing dimethyl sulphoxide and isopropanol from 0 to 80% v/v as additives, in order to obtain an enhancement of the enantioselectivity. For 2-(p-chlorophenoxy)acetic acid and 2-n-butyl-2-(p-chlorophenoxy)acetic acid ethyl esters, DMSO enhanced enzyme enantioselectivity more than IPA with an opposite enzymatic enantiopreference. The cosolvents moderately improved Candida rugosa lipase enantioselectivity for 2-phenyl-2-(p-chlorophenoxy)acetic acid ethyl ester.

Natural Products Syntheses Based on the Biotransformation Using Hydrolases

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Enzymatic ketone reduction: mapping the substrate profile of a short-chain alcohol dehydrogenase (YMR226c) from Saccharomyces cerevisiae

A short-chain alcohol dehydrogenase (YMR226c) from Saccharomyces cerevisiae was cloned and expressed in Escherichia coli, and the encoded protein was purified. The activity and enantioselectivity of this recombinant enzyme were evaluated with a series of ketones. The alcohol dehydrogenase (YMR226c) was found to effectively catalyze the enantioselective reductions of aryl-substituted acetophenones, α-chloroacetophenones, aliphatic ketones, and α- and β-ketoesters. While the enantioselectivity for the reduction of β-ketoesters was moderate, the acetophenone derivatives, aromatic α-ketoesters, some substituted α-chloroacetophenones, and aliphatic ketones were reduced to the corresponding chiral alcohols with excellent enantioselectivity. The enantiopreference of this enzyme generally followed Prelog’s rule for the simple ketones. The ester functionality played some role in determining the enzyme’s enantiopreference for the reduction of α- and β-ketoesters. The present study serves as a valuable guidance for the future applications of this versatile biocatalyst.

Construction of 3D models of the CYP11B family as a tool to predict ligand binding characteristics.

Aldosterone is synthesised by aldosterone synthase (CYP11B2). CYP11B2 has a highly homologous isoform, steroid 11β-hydroxylase (CYP11B1), which is responsible for the biosynthesis of aldosterone precursors and glucocorticoids. To investigate aldosterone biosynthesis and facilitate the search for selective CYP11B2 inhibitors, we constructed three-dimensional models for CYP11B1 and CYP11B2 for both human and rat. The models were constructed based on the crystal structure of Pseudomonas Putida CYP101 and Oryctolagus Cuniculus CYP2C5. Small steric active site differences between the isoforms were found to be the most important determinants for the regioselective steroid synthesis. A possible explanation for these steric differences for the selective synthesis of aldosterone by CYP11B2 is presented. The activities of the known CYP11B inhibitors metyrapone, R-etomidate, R-fadrazole and S-fadrazole were determined using assays of V79MZ cells that express human CYP11B1 and CYP11B2, respectively. By investigating the inhibitors in the human CYP11B models using molecular docking and molecular dynamics simulations we were able to predict a similar trend in potency for the inhibitors as found in the in vitro assays. Importantly, based on the docking and dynamics simulations it is possible to understand the enantioselectivity of the human enzymes for the inhibitor fadrazole, the R-enantiomer being selective for CYP11B2 and the S-enantiomer being selective for CYP11B1.

Chemoenzymatic synthesis of enantiomerically enriched α-hydroxyamides

A study on a chemoenzymatic synthesis of model α-hydroxyamide was performed. Special attention was paid to the optimization of the enzymatic process, both on the selection of enzyme and cosolvent. An intriguing influence of cosolvent on the enantioselectivity of Wheat Germ Lipase and Amano PS Lipase catalyzed hydrolysis was observed, as the results obtained proved that enzyme's enantioselectivity is directly correlated with cosolvent's hydrophobicity. In the best example ( Wheat Germ lipase, Et 2O used as a cosolvent), the reaction proceeded with E = 55, and the target compound was obtained in 33% yield with 92.7%ee.

Nuclear magnetic relaxation of water in ionic‐liquid solutions: determining the kosmotropicity of ionic liquids and its relationship with the enzyme enantioselectivity

AbstractThe nuclear magnetic relaxations of water in ionic‐liquid (IL) solutions were examined at various IL concentrations in order to better understand their hydration behaviors (in terms of nuclear magnetic resonance (NMR) B′‐coefficients). From these B′‐values of ILs, the individual ion's B′‐coefficients were further calculated based on the additivity. These coefficients represent the hydration behavior (‘kosmotropicity’) of ILs in aqueous solutions. Using these data, a linear correlation was found between enzyme enantioselectivity in aqueous solution and the δ′ parameter (difference in NMR B′‐coefficients of anion and cation). In general, high enzyme enantiomeric ratios (E) could be achieved in solutions of ILs with high δ′ values. Copyright © 2007 Society of Chemical Industry

加水分解酵素による物質変換を基盤とした天然物合成

This review summarizes the chemoenzymatic syntheses of biologically active natural products based on a combination of chemical diastereoselectivity and enzymatic enantioselectivity using lipase. Both primary alcohols possessing a chiral center at β-position of hydroxyl group and secondary alcohols were subjected to the lipase-assisted acylation in the presence of acyl donor to afford the optically active esters and the optically active alcohols corresponding to the starting material. These optically active compounds were converted to the biologically active natural products such as bisabolane type sesquiterpenes, oudemansins, nikkomycins B and Z, chuangxinmycin, cystothiazoles and melithiazols possessing antifungal and cytotoxicic activities, inhibition of NADH oxidation, etc.

Inverting enantioselectivity of Burkholderia gladioli esterase EstB by directed and designed evolution

Esterase EstB from Burkholderia gladioli, showing moderate S-enantioselectivity (E(S)=6.1) in the hydrolytic kinetic resolution of methyl-beta-hydroxyisobutyrate, was subjected to directed evolution in order to reverse its enantioselectivity. After one round of ep-PCR, saturation mutagenesis and high-throughput screening, it was found that different mutations at position 152 (in the vicinity of the active site) increase, decrease and even reverse the natural enantioselectivity of this enzyme. The newly created R-enantioselectivity of the esterase mutein (E(Rapp)=1.5) has been further enhanced by a designed evolution strategy involving random mutations close to the active site. Based on the three-dimensional structure nineteen amino acid residues have been selected as mutation sites for saturation mutagenesis. Mutations at three sites (135, 253 and 351) were found to increase R-enantioselectivity. Successive rounds of saturation mutagenesis at these "hot spots" resulted in an increase in R-enantioselectivity from E(Rapp)=1.5 for the parent mutant to E(Rapp)=28.9 for the best variant which carried four amino acid substitutions. Our results prove designed evolution followed by high-throughput screening to be an efficient strategy for engineering enzyme enantioselectivity.

Multicomponent diversity and enzymatic enantioselectivity as a route towards both enantiomers of α-amino acids—a model study

A model study on a new, enantioconvergent method for the synthesis of chiral, nonracemic α-amino acids is presented. α-Acetoxyamides obtained in a Passerini multicomponent reaction are selectively hydrolyzed by Wheat Germ lipase. Studies on conversion of the thus obtained, enantiomerically enriched α-hydroxyamides into α-aminoamides are presented. Products of these reactions are then hydrolyzed to give α-amino acids.

Efficient Chemoenzymatic Synthesis of Pelitrexol via Enzymic Differentiation of a Remote Stereocenter

[structure: see text] An efficient chemoenzymatic process is described for the synthesis of pelitrexol, a novel GARFT inhibitor. The remoteness of this molecule's stereocenter in the tetrahydropterin moiety from the terminal carbonyl group provided a significant challenge in synthesis. The introduction of an oxalamic ester adjacent to the stereocenter dramatically enhanced an enzyme's enantioselectivity for hydrolysis at the terminal ester, producing the desired S-acid with high optical purity and yield. The recycling of the "wrong" enantiomer is achieved via a dehydrogenation/hydrogenation strategy.

The lid is a structural and functional determinant of lipase activity and selectivity

In several lipases access to the enzyme active site is regulated by the position of a mobile structure named the lid. The role of this region in modulating lipase function is reviewed in this paper analysing the results obtained with three different recombinant lipases modified in the lid sequence: Candida rugosa lipase isoform 1 (CRL1), Pseudomonas fragi lipase (PFL) and Bacillus subtilis lipase A (BSLA). A CRL chimera enzyme obtained by replacing its lid with that of another C. rugosa lipase isoform (CRL1LID3) was found to be affected in both activity and enantioselectivity in organic solvent. Variants of the PFL protein in which three polar lid residues were replaced with amino acids strictly conserved in homologous lipases displayed altered chain length preference profile and increased thermostability. On the other hand, insertion of lid structures from structurally homologous enzymes into BSLA, a lipase that naturally does not possess such a lid structure, caused a reduction in the enzyme activity and an altered substrate specificity. These results strongly support the concept that the lid plays an important role in modulating not only activity but also specifity, enantioselectivity and stability of lipase enzymes.

Hofmeister series of ionic liquids: kosmotropic effect of ionic liquids on the enzymatic hydrolysis of enantiomeric phenylalanine methyl ester

The kinetic hydrolysis of enantiomeric phenylalanine methyl ester catalyzed by Bacillus licheniformis protease was performed in aqueous solutions of several hydrophilic ionic liquids (ILs). The protease enantioselectivity was found related to the kosmotropicity of individual cations and anions of ILs. The ion effectiveness in enhancing the enzyme enantioselectivity follows the Hofmeister series: kosmotropic anions and chaotropic cations stabilize the enzyme. In this application, the Hofmeister series of ILs was established in an order of decreasing effectiveness for anions: PO 4 3 - > citrate 3−, CH 3COO −, EtSO 4 - , CF 3COO − > Br − > OTs −, BF 4 - and for cations: [EMIM] + > [BMIM] + > [HMIM] +. The overall IL kosmotropicity was quantified by the δ value (difference in the Jones–Dole viscosity B-coefficients of anion and cation). In general, a high enzyme enantioselectivity was observed in a solution of IL with a high δ value.

Study on the relationship between structure and enantioselectivity of a hyperthermophilic esterase from archaeon Aeropyrum pernix K1

To enhance the enantioselectivity of a hyperthermophilic esterase from archaeon Aeropyrum pernix K1 (APE1547), a directed evolution approach is employed to generate mutant library from the native enzyme. A mutation (TBC26) is identified after one round of epPCR. The enantioselectivity of TBC26 is increased up to 2.6-fold compared to that of wild type enzyme. TBC26 contains five amino acid substitutions (R11G, L36P, V223A, I551L, A564T). The five mutation sites are spatially distant to the catalytic center. According to the published crystal structure of WT and considering the changes of secondary and tertiary structure, here we try to explain the change of enantioselectivity of the TBC26. The results suggest that the change of enantioselectivity of enzyme has a close relationship to the configuration of the enzyme.

Enhancement of enantioselectivity in lipase-catalyzed resolution of N-(2-ethyl-6-methylphenyl)alanine by additives

The lipase B from Candida antarctica (CAL-B)-catalyzed the enantioselective resolution of N-(2-ethyl-6-methylphenyl)alanine from the corresponding racemic methyl ester is investigated. However, the enantioselectivity of CAL-B towards the resolution is not high enough to obtain enantiomerically pure compound. With the aim to improve the enantioselectivity of enzyme, various additives including macrocyclic dioxotetraamines, organic compounds and ionic liquids are tested in the CAL-B-catalyzed hydrolysis. Under the optimized conditions, the highest enantioselectivity of CAL-B ( E > 100) is achieved in diethyl ether/water (15%, v/v), which is about 9.7-fold more enantioselective than that in pure buffered aqueous solution ( E = 12.1). When the macrocylic dioxotetraamine ( n-C 6H 13-MDTA) is added in the reaction medium, both catalytic activity and enantioselectivity of CAL-B are markedly enhanced ( E-value is up to 87.6 at 45.6% conversion only in 1.5 h). While the E-value is improved only up to 44.7 using ionic liquid ([ETOMG]BF 4) as additive, it can establish the green environment of enzymatic catalysis.

A validated gas chromatographic method for the evaluation of enzymatic enantioselectivity in kinetic resolution applications

An enantioselective gas chromatography (GC) method has been developed and validated for determination of the enantiomers of citronellol in kinetic resolution experiments. S-(-)-beta-Citronellol is a precursor of rose oxide. After solid-phase extraction (SPE) with ethyl acetate, the enantiomers of R-(+)-beta-citronellol and S-(-)-beta-citronellol and their corresponding acetate- and butyrate esters were separated through enantioselective GC respectively. The method was validated and found to be reproducible, specific, accurate, and precise. Analyte recoveries and detection limits were also determined. The applicability of this method was shown in a kinetic resolution experiment using lipase A of Bacillus subtilis.

Enhancement of enzyme activity and enantioselectivity by cyclopentyl methyl ether in the transesterification catalyzed by Pseudomonas cepacia lipase co-lyophilized with cyclodextrins

The solvent effects of cyclopentyl methyl ether (CPME) on the reaction rates and enzyme enantioselectivity in the enantioselective transesterifications of racemic 6-methyl-5-hepten-2-ol (racemic sulcatol: SUL) and racemic 2,2-dimethyl-1,3-dioxolane-4-methanol (racemic solketal: SOL) with a series of enol esters catalyzed by Pseudomonas cepacia lipase co-lyophilized with cyclodextrins (alpha-, beta-, gamma-, partially methylated beta-, and 2,3,6-tri-O-methyl-beta-cyclodextrin: alphaCyD; betaCyD; gammaCyD; Me1.78betaCyD; Me3betaCyD) were investigated and compared with those in diisopropyl ether (IPE). In the case of SUL, enzyme activities of the co-lyophilizate with Me1.78betaCyD in CPME were lower than those in IPE with every acyl source, however, the absolute enantiopreference was shown in the transesterification with vinyl butyrate (VBR) in IPME. When the substrates were SOL and VBR, the enzyme activities in CPME were greatly enhanced as high as 1.6-9.8-fold, while the enantioselectivities in CPME were comparable to those in IPE.

Molecular design of two sterol 14α-demethylase homology models and their interactions with the azole antifungals ketoconazole and bifonazole

Sterol 14alpha-demethylase (CYP51) is one of the known major targets for azole antifungals. Therapeutic side effects of these antifungals are based on interactions of the azoles with the human analogue enzyme. This study describes for the first time a comparison of a human CYP51 (HU-CYP51) homology model with a homology model of the fungal CYP51 of Candida albicans (CA-CYP51). Both models are constructed by using the crystal structure of Mycobacterium tuberculosis MT-CYP51 (PDB code: 1EA1). The binding mode of the azole ketoconazole is investigated in molecular dynamics simulations with the GROMACS force field. The usage of special parameters for the iron azole complex binding is necessary to obtain the correct complex geometry in the active site of the enzyme models. Based on the dynamics simulations it is possible to explain the enantioselectivity of the human enzyme and also to predict the binding mode of the isomers of ketoconazole in the active site of the fungal model.