Enantioselectivity Of Lipase Research Articles

ChemInform Abstract: Effects of Medium and of Reaction Conditions on the Enantioselectivity of Lipases in Organic Solvents and Possible Rationales.

Abstract The nature of organic solvents markedly influenced the enantioselectivity, expressed as the enantiomeric ratio, of porcine pancreatic lipase and of Lipase PS in transesterification between trifluoroethyl butanoate and the two unrelated alcohols (±)-sulcatol ( 1 ) and (±)-3-bromo-5-hydroxymethyl isoxazoline ( 2 ). However, there was no correlation between enantioselectivity and such physicochemical characteristics of the solvent as hydrophobicity and dielectric constant. A rationale based on the formation of solvent-enzyme complexes is proposed to explain the results. Enzyme enantioselectivity was also affected by temperature but not by the chain length of the acylating agent nor by the removal of water from the reaction medium by molecular sieves. The effects of these parameters on transesterification rates were also investigated.

Significantly Improved Expression and Biochemical Properties of Recombinant Serratia marcescens Lipase as Robust Biocatalyst for Kinetic Resolution of Chiral Ester

A lipase gene from Serratia marcescens ECU1010 was cloned into expression vector pET28a, sequenced, and overexpressed as an N terminus His-tag fusion protein in Escherichia coli. Through the optimization of culture conditions in shake flask, the lipase activity was improved up to 1.09 x 10⁵ U/l, which is a great improvement compared to our previous reports. It was purified to homogeneity by Ni-NTA affinity chromatography with an overall yield of 59.4% and a purification factor of 2.4-fold. This recombinant lipase displayed excellent stability below 30 °C and within the pH range of 5.0-6.8, giving temperature and pH optima at 40 °C and pH 9.0, respectively. The lipase activity was found to increase in the presence of metal ions such as Ca²+, Cu²+, and some nonionic surfactants such as PEG series. In addition, among p-nitrophenyl esters of fatty acids with varied chain length, the recombinant lipase showed the maximum activity on p-nitrophenyl laurate (C₁₂). Using racemic trans-3-(4'-methoxy-phenyl)-glycidyl methyl ester [(±)-MPGM] as substrate, which is a key chiral synthon for production of diltiazem, a 50% conversion yield was achieved after 4 h in toluene-water (100 mM KPB phosphate buffer, pH 7.5) biphasic system (5:5 ml) at 30 °C under shaking condition (160 rpm), affording (-)-MPGM in nearly 100% ee. The K(m) and V(max) values of the lipase for (±)-MPGM were 222 mM and 1.24 mmol min⁻¹ mg⁻¹, respectively. The above-mentioned features make the highly enantioselective lipase from Serratia marcescens ECU1010 a robust biocatalyst for practical use in large-scale production of diltiazem intermediate.

Directed Evolution of an Enantioselective Lipase with Broad Substrate Scope for Hydrolysis of α-Substituted Esters

A variant of Candida antarctica lipase A (CalA) was developed for the hydrolysis of alpha-substituted p-nitrophenyl esters by directed evolution. The E values of this variant for 7 different esters was 45-276, which is a large improvement compared to 2-20 for the wild type. The broad substrate scope of this enzyme variant is of synthetic use, and hydrolysis of the tested substrates proceeded with an enantiomeric excess between 95-99%. A 30-fold increase in activity was also observed for most substrates. The developed enzyme variant shows (R)-selectivity, which is reversed compared to the wild type that is (S)-selective for most substrates.

Kinetic resolution of (R,S)-pyrazolides containing substituents in the leaving pyrazole for increased lipase enantioselectivity

Abstract With hydrolysis of ( R , S )-azolides in water-saturated methyl tert -butyl ether (MTBE) via Candida antarctica lipase B (CALB) as the model system, ( R , S )-pyrazolides containing a leaving 3-, 4- or 3,4-substituted-pyrazole moiety are selected as the best substrates for preparing various optically pure carboxylic acids containing an α-chiral center. Great improvements of enzyme activity for the ( R )-enantiomers with excellent enantioselectivity ( V R / V S > 100) are obtainable, if ( R , S )-pyrazolides containing a leaving 3- or 3,4-substituted-pyrazole moiety are employed for the hydrolysis or alcoholysis by methanol in anhydrous MTBE. A detailed kinetic analysis for ( R , S )- N -2-phenylpropionylpyrazoles indicates that a bulky 3-substituent such as 3-(3-bromophenyl) or 3-(2-pyridyl) in the leaving pyrazole moiety has profound effects on decreasing the nucleophilic attack and proton transfer of catalytic serine for the slow-reacting enantiomer in anhydrous MTBE, as well as that and substrate affinity for both enantiomers in water-saturated MTBE. The resolution platform is also successfully applied to the hydrolysis of ( R , S )-pyrazolides in water-saturated cyclohexane via Candida rugosa lipase (Lipase MY) having opposite enantioselectivity to CALB.

Rational creation of mutant enzyme showing remarkable enhancement of catalytic activity and enantioselectivity toward poor substrates

Catalytic activity and enantioselectivity of lipase toward poor substrates bearing bulky substituents on both sides have been dramatically improved by rational design; the E value for a poor substrate was increased from 5 (wild-type enzyme) to >200 (I287F/I290A double mutant) with an acceleration of the reaction rate.

Prediction of the enantioselectivity of lipases and esterases by molecular docking method with modified force field parameters

In the work, molecular docking method was applied to extensively predict the enantioselectivity of lipases and esterases. A ligand library consisted of 69 chiral substrates was docked to four lipases and two esterases to set up the prediction model. During the docking process, necessary modification was carried out on van de Waals and hydrogen bond parameters of enzyme/substrate pair so that the ligands were able to adopt productive geometry in the enzymes. The docking results correctly indicated the enantiopreference for 91% (63/69) of docking pairs and the docking energy difference between substrate enantiomers (Delta Delta G(docking)) was significantly (correlation coefficient = 0.72, P < 0.05) correlated with the activation free energy difference (Delta Delta G( not equal)) that was quantitatively correlated with enantioselectivity of the enzymes. The prediction method was further validated by docking with another 12 enzyme/substrate pairs. Moreover, the prediction error was susceptible to the size of groups bonded to substrate's chiral center and expected Delta Delta G( not equal) values but was not related to the substrate type and reaction medium. The possible reasons of observed error were discussed. It is demonstrated that the docking method has great application potential in high performance prediction of enzyme enantioselectivity.

Disparity in productive binding mode of the slow-reacting enantiomer determines the novel catalytic behavior of Candida antarctica lipase B

In the context of specifying the origin of enzyme enantioselectivity, the present study explores the lipase enantioselectivity towards secondary alcohols of similar structure from the perspective of substrate binding. By carrying out molecular mechanics minimization as well as molecular dynamics simulation on tetrahedral reaction intermediates which are used as a model of transition state, we identify an unconventional productive binding mode (PBM)—M/H permutation type for Candida antarctica lipase B (CALB). The in silico results also indicate that different PBMs of the slow-reacting enantiomer do exist in one lipase even when there is little structural differences between substrates, e.g. compounds with Ph or CH 2CH 2Ph group display the M/H permutation type PBM while molecules with CH 2Ph show the M/L permutation type PBM. By relating the PBMs of substrates to the experimentally determined E-values obtained by Hoff et al. [16], we find that disparity in PBM of the slow-reacting enantiomer determines why E-values of substrates with CH 2Ph were lower than E-values of substrates with Ph or CH 2CH 2Ph group. The modeling results also suggest that the “pushed aside” effect of the F atom and Br atom accommodates the medium size substituent of the substrate better in the stereospecificity pocket of the enzyme.

Cover Picture: Control of Lipase Enantioselectivity by Engineering the Substrate Binding Site and Access Channel (ChemBioChem 17/2009)

Cover Picture: Control of Lipase Enantioselectivity by Engineering the Substrate Binding Site and Access Channel (ChemBioChem 17/2009)

Control of Lipase Enantioselectivity by Engineering the Substrate Binding Site and Access Channel

Lipase from Burkholderia cepacia (BCL) has proven to be a very useful biocatalyst for the resolution of 2-substituted racemic acid derivatives, which are important chiral building blocks. Our previous work showed that enantioselectivity of the wild-type BCL could be improved by chemical engineering of the substrate's molecular structure. From this earlier study, three amino acids (L17, V266 and L287) were proposed as targets for mutagenesis aimed at tailoring enzyme enantioselectivity. In the present work, a small library of 57 BCL single mutants targeted on these three residues was constructed and screened for enantioselectivity towards (R,S)-2-chloro ethyl 2-bromophenylacetate. This led to the fast isolation of three single mutants with a remarkable tenfold enhanced or reversed enantioselectivity. Analysis of substrate docking and access trajectories in the active site was then performed. From this analysis, the construction of 13 double mutants was proposed. Among them, an outstanding improved mutant of BCL was isolated that showed an E value of 178 and a 15-fold enhanced specific activity compared to the parental enzyme; thus, this study demonstrates the efficiency of the semirational engineering strategy.

A High-Throughput Screening Method for Chiral Alcohols and its Application to Determine Enantioselectivity of Lipases and Esterases

A new colorimetric method to determine the enantiopurity of chiral alcohols is presented based on enantioselective oxidation by two alcohol dehydrogenases. The oxidation of either (R)- or (S)-alcohol produces NADPH (or NADH), which reduces 2-(4-iodophenyl)-3-(4- nitrophenyl)-5-phenyl-2H-tetrazolium to its corresponding formazan derivative. Application of this method to screen lipase variant libraries for enantioselective hydrolysis is also described.

Response surface analysis for the production of an enantioselective lipase from Aspergillus niger by solid-state fermentation

The lipase produced by the Aspergillus niger strain AC-54 has been widely studied due to its enantioselectivity for racemic mixtures. This study aimed to optimize the production of this enzyme using statistical methodology. Initially a Plackett-Burman (PB) design was used to evaluate the effects of the culture medium components and the culture conditions. Twelve factors were screened: water content, glucose, yeast extract, peptone, olive oil, temperature, NaH(2)P0(4), KH(2)P0(4), MgS0(4)-7H(2)0, CaCl(2), NaCI, and MnS0(4). The screening showed that the significant factors were water content, glucose, yeast extract, peptone, NaH(2)P0(4), and KH(2)P0(4), which were optimized using response surface methodology (RSM) and a mathematical model obtained to explain the behavioral process. The best lipase activity was attained using the following conditions: water content (20%), glucose (4.8%), yeast extract (4.0%), and NaH2P04 (4.0%). The predicted lipase activity was 33.03 U/ml and the experimental data confirmed the validity of the model. The enzymatic activity was expressed as micromoles of oleic acid released per minute of reaction (micromol/min).

Burkholderia cepacia lipase is an excellent enzyme for the enantioselective hydrolysis of β-heteroaryl-β-amino esters

The enantioselective ( E >200) lipase PS-catalysed hydrolysis of β-heteroaryl-β-amino esters is described. The reactions were performed with H 2O (0.5 equiv) in either diisopropyl ether or tert-butyl methyl ether at 25 °C. The resulting β-heteroaryl-substituted β-amino acid enantiomers were formed in high enantiomeric excess (ee ⩾ 97%) and in good yield (⩾40%).

Increased enantioselectivity of lipase in the transesterification of dl-(±)-3-phenyllactic acid in ionic liquids

Three lipases from different sources (Pseudomonas aeruginosa MTCC 5113 (PAL), porcine pancreatic (PPL) and Candida rugosa (CRL)) were screened for the transesterification of dl-3-phenyllactic acid (dl-3-PLA) in three different ionic liquids (1-butyl-3-methyl imidazolium tetrafluoroborate ([BMIM][BF4]), 1-butyl-3-methyl imidazolium hexafluorophosphate ([BMIM][PF6]) and 1-ethyl-3-methyl imidazolium tetrafluoroborate ([EMIM][BF4])) in combination with different organic solvents (e.g. hexane, cyclohexane, isooctane, toluene, vinyl acetate and dichloromethane). CRL efficiently catalyzed the esterification of dl-3-PLA to l-O-acetyl-3-PLA using vinyl acetate as acyl donor, showing excellent enantioselectivity (E~100) and conversion in all three ionic liquids. PPL exhibited enantioselectivity only in the presence of [BMIM][BF4] and was not able to catalyze dl-3-PLA esterification in the other two ionic liquids. PAL did not show any reactivity with dl-3-PLA in any of the ionic liquids. Various parameters such as solvent and ionic liquid concentration, reaction time, substrate and enzyme concentration on the activity and enantioselectivity of CRL were studied.

Improvement of Yarrowia lipolytica Lipase Enantioselectivity by Using Mutagenesis Targeted to the Substrate Binding Site

Lip2p lipase from Yarrowia lipolytica was shown to be an efficient catalyst for the resolution of 2-bromo-arylacetic acid esters, an important class of chemical intermediates in the pharmaceutical industry. Enantioselectivity of this lipase was improved by site-directed mutagenesis targeted to the substrate binding site. To guide mutagenesis experiments, the three-dimensional model of this lipase was built by homology modelling techniques by using the structures of lipases from Rhizomucor miehei and Thermomyces lanuginosa as templates. On the basis of this structural model, five amino acid residues (T88, V94, D97, V232, V285) that form the hydrophobic substrate binding site of the lipase were selected for site-directed mutagenesis. Position 232 was identified as crucial for the discrimination between enantiomers. Variant V232A displayed an enantioselectivity enhanced by one order of magnitude, whereas variant V232L exhibited a selectivity inversion. To further explore the diversity, position 232 was systematically replaced by the 19 possible amino acids. Screening of this library led to the identification of the V232S variant, which has a tremendously increased E value compared to the parental enzyme for the resolution of 2-bromo-phenylacetic acid ethyl ester (58-fold) and 2-bromo-o-tolylacetic acid ethyl ester (16-fold). In addition to the gain in enantioselectivity, a remarkable increase in velocity was observed (eightfold increase) for both substrates.

Modelling substrate specificity and enantioselectivity for lipases and esterases by substrate-imprinted docking

BackgroundPreviously, ways to adapt docking programs that were developed for modelling inhibitor-receptor interaction have been explored. Two main issues were discussed. First, when trying to model catalysis a reaction intermediate of the substrate is expected to provide more valid information than the ground state of the substrate. Second, the incorporation of protein flexibility is essential for reliable predictions.ResultsHere we present a predictive and robust method to model substrate specificity and enantioselectivity of lipases and esterases that uses reaction intermediates and incorporates protein flexibility. Substrate-imprinted docking starts with covalent docking of reaction intermediates, followed by geometry optimisation of the resulting enzyme-substrate complex. After a second round of docking the same substrate into the geometry-optimised structures, productive poses are identified by geometric filter criteria and ranked by their docking scores. Substrate-imprinted docking was applied in order to model (i) enantioselectivity of Candida antarctica lipase B and a W104A mutant, (ii) enantioselectivity and substrate specificity of Candida rugosa lipase and Burkholderia cepacia lipase, and (iii) substrate specificity of an acetyl- and a butyrylcholine esterase toward the substrates acetyl- and butyrylcholine.ConclusionThe experimentally observed differences in selectivity and specificity of the enzymes were reproduced with an accuracy of 81%. The method was robust toward small differences in initial structures (different crystallisation conditions or a co-crystallised ligand), although large displacements of catalytic residues often resulted in substrate poses that did not pass the geometric filter criteria.

A Three‐Dimensional Quanititative Structure‐Activity Relationship (3D‐QSAR) Model for Predicting the Enantioselectivity of Candida antarctica Lipase B

Abstractmagnified imageComputational techniques involving molecular modeling coupled with multivariate statistical analysis were used to evaluate and predict quantitatively the enantioselectivity of lipase B from Candida antarctica (CALB). In order to allow the mathematical and statistical processing of the experimental data largely available in the literature (namely enantiomeric ratio E), a novel class of GRID‐based molecular descriptors was developed (differential molecular interaction fields or DMIFs). These descriptors proved to be efficient in providing the structural information needed for computing the regression model. Multivariate statistical methods based on PLS (partial least square – projection to latent structures), were used for the analysis of data available from the literature and for the construction of the first three‐dimensional quanititative structure‐activity relationship (3D‐QSAR) model able to predict the enantioselectivity of CALB. Our results indicate that the model is statistically robust and predictive.

Enantioselective behavior of lipases from Aspergillus niger immobilized in different supports

Considering the extraordinary microbial diversity and importance of fungi as enzyme producers, the search for new biocatalysts with special characteristics and possible applications in biocatalysis is of great interest. Here, we report the performance in the resolution of racemic ibuprofen of a native enantioselective lipase from Aspergillus niger, free and immobilized in five types of support (Accurel EP-100, Amberlite MB-1, Celite, Montmorillonite K10 and Silica gel). Amberlite MB-1 was found to be the best support, with a conversion of 38.2%, enantiomeric excess of 50.7% and enantiomeric ratio (E value) of 19 in 72 h of reaction. After a thorough optimization of several parameters, the E value of the immobilized Aspergillus niger lipase was increased (E = 23) in a shorter reaction period (48 h) at 35 degrees C. Moreover, the immobilized Aspergillus niger lipase maintained an esterification activity of at least 80% after 8 months of storage at 4 degrees C and could be reused at least six times.

Improvement of the enantioselectivity and activity of lipase from Pseudomonas sp. via adsorption on a hydrophobic support: kinetic resolution of 2-octanol

Improvement of the enantioselectivity and activity of lipase from Pseudomonas sp. via adsorption on a hydrophobic support: kinetic resolution of 2-octanol

Improvement of the enantioselectivity and activity of lipase from Pseudomonas sp. via adsorption on a hydrophobic support: kinetic resolution of 2-octanol

Pseudomonas sp. lipase (PSL) was successfully immobilized on a novel hydrophobic polymer support through physical adsorption and the immobilized PSL was used for resolution of (R,S)-2-octanol with vinyl acetate as acyl donor. Enhanced activity and enantioselectivity were observed from the immobilized PSL compared with free PSL. The effects of reaction conditions such as temperature, water activity, substrate molar ratio and the amount of immobilized lipase were investigated. Under optimum conditions, the residual (S)-2-octanol was recovered with 99.5% enantiomeric excess at 52.9% conversion. The results also indicated that the immobilized PSL could maintain 94% of its initial activity even after reusing it five times.

Kinetic resolution of 1‐phenylethanol integrated with separation of substrates and products by a supported ionic liquid membrane

AbstractBACKGROUND: In the present study, the kinetic resolution of rac‐1‐phenylethanol by transesterification with several vinyl esters catalysed by a commercial immobilized Candida antarctica lipase B (Novozym 435) was carried out in n‐hexane at different water contents. The subtrates and products involved in the kinetic resolution were separated using a membrane bioreactor containing a supported liquid membrane based on the ionic liquid 1‐butyl‐3‐methylimidazolium tetrafluoroborate [bmim+][BF4−].RESULTS: Variables affecting the kinetic resolution performance of the enzyme were studied. First, the influence of water content of the medium on the synthetic activity, selectivity and enantioselectivity of the enzyme was analysed in order to establish the optimal amount of water. The use of vinyl esters of different alkyl chain length (vinyl propionate, vinyl butyrate and vinyl laurate) as acyl donors to kinetic resolution was studied. Finally, the integrated reaction/separation process for the resolution of rac‐1‐phenylethanol was carried out in the optimal conditions found.CONCLUSION: These investigations demonstrate that the coupling of lipase enantioselectivity with the selective separation of supported liquid membranes based on ionic liquids provides a promising basis for practical production of enantiomerically pure or enriched compounds. Copyright © 2008 Society of Chemical Industry