Selectivity-Enhancement of Hydrolase Reactions

This review pinpoints the strategies which can be employed to improve the enantio- and diasteroselectivity of hydrolytic enzymes, i.e. esterases, proteases, and lipases. The influence of variations of reactants, — enzyme and substrate — and conditions — kinetics, medium, temperature, pH — on the chiral recognition process of the enzyme is discussed with examples from the recent literature.     

Biochemical profiling in silico--predicting substrate specificities of large enzyme families

A general high-throughput method for in silico biochemical profiling of enzyme families has been developed based on covalent docking of potential substrates into the binding sites of target enzymes. The method has been tested by systematically docking transition state--analogous intermediates of 12 substrates into the binding sites of 20 alpha/beta hydrolases from 15 homologous families. To evaluate the effect of side chain orientations to the docking results, 137 crystal structures were included in the analysis. A good substrate must fulfil two criteria: it must bind in a productive geometry with four hydrogen bonds between the substrate and the catalytic histidine and the oxyanion hole, and a high affinity of the enzyme-substrate complex as predicted by a high docking score. The modelling results in general reproduce experimental data on substrate specificity and stereoselectivity: the differences in substrate specificity of cholinesterases toward acetyl- and butyrylcholine, the changes of activity of lipases and esterases upon the size of the acid moieties, activity of lipases and esterases toward tertiary alcohols, and the stereopreference of lipases and esterases toward chiral secondary alcohols. Rigidity of the docking procedure was the major reason for false positive and false negative predictions, as the geometry of the complex and docking score may sensitively depend on the orientation of individual side chains. Therefore, appropriate structures have to be identified. In silico biochemical profiling provides a time efficient and cost saving protocol for virtual screening to identify the potential substrates of the members of large enzyme family from a library of molecules.     

Cloning,screening and characterization of ester hydrolases with enantioselectivity in typical bacteria

A broad exploitation of ester hydrolases from 7 typical bacteria was reported in this study. Thirty-two predicted esterases and hydrolases were cloned based on published genomic information. The catalytic activity of obtained clones was tested with p-nitrophenyl esters at various temperatures and pH values. The results indicated that eight enzymes presented with typical esterase activity on p-nitrophenyl butylate and caprylate. The result also showed that despite their great sequence difference, the eight enzymes shared similar properties (substrate specificity, optimal pH and temperature) with each other. Phylogenetic analysis revealed a close relationship between these eight enzymes and “true esterases”. As there was no information on enantioselectivity of these enzymes reported, the enantioselectivity of these enzymes to various chiral substrates was investigated for the first time. In comparison with commercial enzyme, Candida rugosa lipase (CRL), enzymes E12, E14, E18, E21 and E24 presented with equal or higher activity and enantioselectivity to the substrates. Furthermore, enzyme E14 (predicted carboxylesterase from Rhodobacter sphaeroides), E21 (S-formylglutathione hydrolase from Pseudomonas putida) and E24 (carboxylesterase from P. putida) presented with enantioselectivity in the resolution of methyl mandelate, 1-phenyethyl acetate and 2-octanol. These findings suggested that the novel ester hydrolases with high activity and enantioselectivity could be obtained from alpha/beta hydrolase family.     

Selectivity-Enhancement of Hydrolase Reactions

This review pinpoints the strategies which can be employed to improve the enantio- and diasteroselectivity of hydrolytic enzymes, i.e. esterases, proteases, and lipases. The influence of variations of reactants, — enzyme and substrate — and conditions — kinetics, medium, temperature, pH — on the chiral recognition process of the enzyme is discussed with examples from the recent literature.     

Kinetic and thermodynamic properties of purified alkaline protease from Bacillus pumilus Y7 and non‐covalent immobilization to poly(vinylimidazole)/clay hydrogel

The characterization of the hydrogel was performed using Fourier‐transform infrared spectroscopy, X‐ray diffraction, and scanning electron microscopy. Purified Bacillus pumilus Y7‐derived alkaline protease was immobilized in Poly (vinylimidazole)/clay (PVI/SEP) hydrogel with 95% yield of immobilization. Immobilization decreased the pH optimum from 9 to 6 for free and immobilized enzyme, respectively. Temperature optimum 3°C decreased for immobilized enzyme. The K_m, V_m, and k_cat of immobilized enzyme were 4.4, 1.7, and 7.5‐fold increased over its free counterpart. Immobilized protease retained about 65% residual activity for 16^th reuse. The immobilized protease endured its 35% residual activity in the material after six cycle's batch applications. The results of thermodynamic analysis for casein hydrolysis showed that the ΔG^≠ (activation free energy) and ΔG^≠_E‐T (activation free energy of transition state formation) obtained for the immobilized enzyme decreased in comparison to those obtained for the free enzyme. On the other hand, the value of ΔG^≠_ES (free energy of substrate binding) was observed to have increased. These results indicate an increase in the spontaneity of the biochemical reaction post immobilization. Enthalpy value of immobilized enzyme that was 2.2‐fold increased over the free enzyme indicated lower energy for the formation of the transition state, and increased ΔS^≠ value implied that the immobilized form of the enzyme was more ordered than its free form.     

Release of short chain fatty acids from cream lipids by commercial lipases and esterases

Lipases and esterases are frequently used in dairy production processes to enhance the buttery flavour of the end product. Short chain fatty acids, and especially butanoic acid, play a key role in this and different enzymes with specificity towards short chain fatty acids are commercially available as potent flavouring tools. We have compared six lipases/esterases associated with buttery flavour production. Although specificity to short chain fatty acids was ascribed to each enzyme, clear differences in free fatty acid profiles were found when these enzymes were applied on cream. Candida cylindraceae lipase was the most useful enzyme for buttery flavour production in cream with the highest yield of free fatty acids (57 g oleic acid 100 g⁻¹ fat), no release of long chain fatty acids and specificity towards butanoic acid.     

Kinetic resolution of 2-chloro-3,3,3-trifluoropropanoic acid esters catalyzed by lipase from Candida rugosa

Abstract

By screening around 30 commercially available lipases and esterases, two enzymes, C. rugosa lipase and P. fluorescens esterase, were found to posess catalytic activity and enantioselectivity (E?10) for the hydrolysis of 2-chloro-3,3,3-trifluoropropanoic acid (CTFPA) methyl and ethyl ester. Both enzymes were tentatively assigned to be (S)-selective based on the assumption that they have the same stereopreference as in the hydrolysis of methyl 2-chloropropanoate, which is a non-fluorinated analogue of CTFPA. The enzymes were applied in the kinetic resolution of CTFPA ethyl ester and 95% ee of the remaining ester could be achieved at 60% conversion. The crosslinked enzyme aggregate (CLEA) of C. rugosa lipase was found to catalyze enantioselective transesterification (E?40) of CTFPA methyl ester with ethanol. By conducting the transesterification in a 10-mL packed-bed reactor containing CLEA, it was possible to convert racemic CTFPA methyl ester into the mixture of (S)-methyl and (R)-ethyl esters with 82% and 90% ee, respectively, at 4.0 g/L^-1/h^-1 space-time yield, which decreased to 1.0 g/L^-1/h^-1 after four repetitive batches.     

Physico-chemical properties of the epoxide hydrolase from Rhodosporidium toruloides

Residue-specific chemical modification of amino acid residues of the microsomal epoxide hydrolase (mEH) from Rhodosporidium toruloides UOFS Y-0471 revealed that the enzyme is inactivated through modification of Asp/Glu and His residues, as well as through modification of Ser. Since Asp acts as the nucleophile, and Asp/Glu and His serve as charge relay partners in the catalytic triad of microsomal and soluble epoxide hydrolases during epoxide hydrolysis, inactivation of the enzyme by modification of the Asp/Glu and His residues agrees with the established reaction mechanism of these enzymes. However, the inactivation of the enzyme through modification of Ser residues is unexpected, suggesting that a Ser in the catalytic site is indispensable for substrate binding by analogy of the role of Ser residues in the related L-2-haloacid dehalogenases, as well as the ATPase and phosphatase enzymes. Co²⁺, Hg²⁺, Ag⁺, Mg²⁺ and Ca²⁺ inhibited enzyme activity and EDTA increased enzyme activity. The activation energy for inactivation of the enzyme was 167 kJ mol^–1. Kinetic constants for the enzyme could not be determined since unusual behaviour was displayed during hydrolysis of 1,2-epoxyoctane by the purified enzyme. Enantioselectivity w as strongly dependent on substrate concentration. When the substrate was added in concentrations ensuring two-phase conditions, the enantioselectivity was greatly enhanced. On the basis of these results, it is proposed that this enzyme acts at an interface, analogous to lipases.     

Functional characterization of hypothetical proteins of Mycobacterium tuberculosis with possible esterase/lipase signature: a cumulative in silico and in vitro approach

The functional aspect of several mycobacterium proteins annotated as hypothetical are yet to be discovered. In the present investigation, in silico approaches were used to predict the biological function of some of the unknown Mtb proteins, which were further validated by wet lab experiments. After screening thousands of Mtb proteins, functionally unknown hypothetical proteins Rv0421c, Rv0519c, Rv0774c, Rv1191, Rv1592c, and Rv3591c were chosen on the basis of their importance in Mtb life cycle. All these proteins posses the α/β-hydrolase topological fold, characteristic of lipases/esterases, with serine, aspartate, and histidine as the putative members of the catalytic triad. The catalytic serine is located in pentapeptide motif “GXSXG” and oxyanion residue is in dipeptide motif HG. To further support our observation, molecular docking was performed with conventional synthetic lipolytic substrates (pNP-esterss) and specific lipase/esterase inhibitors (tetrahydrolipstatin and phenylmethanesulfonyl fluoride (PMSF)). Significant docking score and strong interaction of substrates/inhibitors with these proteins revealed that these could be possible lipases/esterases. To validate the in silico studies, these genes were cloned from Mtb genome and the proteins were over-expressed in pQE-30/Escherichia coli M15 system. The expressed proteins were purified to homogeneity and enzymatic activity was determined using pNP esters as substrate. The enzyme activity of recombinant proteins was inhibited by tetrahydrolipstatin and PMSF pre-treatment. Outcome of the present investigation provided a basic platform to analyze and characterize unknown hypothetical proteins.     

A multisubstrate assay for lipases/esterases: Assessing acyl chain length selectivity by reverse-phase high-performance liquid chromatography

Lipases and esterases are hydrolytic enzymes and are known to hydrolyze esters with unique substrate specificity and acyl chain length selectivity. We have developed a simple competitive multiple substrate assay for determination of acyl chain length selectivity of lipases/esterases using RP-HPLC with UV detection. A method for separation and quantification of 4-nitrophenyl fatty acid esters (C₄–C₁₈) was developed and validated. The chain length selectivity of five lipases and two esterases was determined in a multisubstrate reaction system containing equimolar concentrations of 4-nitrophenyl esters (C₄–C₁₈). This assay is simple, reproducible, and a useful tool for determining chain length selectivity of lipases/esterases.     

The structure of a thermally stable 3-phosphoglycerate kinase and a comparison with its mesophilic equivalent

The structure of the phosphoglycerate kinase (PGK) from Bacillus stearothermophilus, a moderate thermophile, has been determined and compared with that of its mesophilic equivalent from yeast. The Bacillus enzyme structure was solved by molecular replacement and improved using constrained rigid-body, molecular dynamics and conventional refinement procedures. The refinement residual, calculated using all the measured data between 8 and 1.65 Å, is 0.18(1). The stereo chemical deviations of the final model from ideality are 0.01 Å for both bonds and planes. The mid-point temperatures of the Bacillus and yeast enzymes are 67 and 53°C, respectively. Differential scanning calorimetry indicates that the energy difference (ΔΔG) between the mesophilic and thermophilic enzymes is of the order of 5 kcal mol^?1 at room temperature. The structure comparison indicates that the features most likely to be responsible for the increased thermal stability of the Bacillus enzyme are the increased internal hydrophobicity, additional ion pairs, and better α-helix stability resulting from the removal of helix destablising residues and extra helix–dipole/helix side chain ionic interactions. © 1993 Wiley-Liss, Inc.     

Isolation and characterization of cold-active family VIII esterases from an arctic soil metagenome

Functional screening for lipolytic enzymes at low temperatures resulted in the isolation of the novel cold-active esterases, EstM-N1 and EstM-N2, from a metagenomic DNA library of arctic soil samples. EstM-N1 and EstM-N2 were 395 and 407 amino acids in length, respectively, and showed the highest similarity to class C β-lactamases. However, they shared a relatively low level of sequence similarity (30%) with each other. Phylogenetic analysis of bacterial lipolytic enzymes confirmed that EstM-N1 and EstM-N2 belonged to family VIII of bacterial esterases/lipases. The (His)₆-tagged esterases were purified to about 99% homogeneity from the soluble fraction of recombinant Escherichia coli cultures. The purified EstM-N1 and EstM-N2 retained more than 50% of maximal activity in the temperature range of 0–35°C, with optimal temperatures of 20°C and 30°C, respectively. Both enzymes preferred the short acyl chains of p-nitrophenyl esters and exhibited very narrow substrate specificity, indicating that they are typical esterases. The β-lactamase activity of EstM-N1 and EstM-N2 was also detected and reached about 31% and 13% of the positive control enzyme, Bacillus cereus β-lactamase, respectively. These first cold-active esterases belonging to family VIII are expected to be useful for potential biotechnological applications as interesting biocatalysts.     

Production of extracellular enzymes by different isolates of Pochonia chlamydosporia

For the first time, the specific activities of chitinases, esterases, lipases and a serine protease (VCP1) produced by different isolates of the nematophagous fungus Pochonia chlamydosporia were quantified and compared. The isolates were grown for different time periods in a minimal liquid medium or media supplemented with 1 % chitin, 0.2 % gelatin or 2 % olive oil. Enzyme-specific activities were quantified in filtered culture supernatants using chromogenic p-nitrophenyl substrates (for chitinases, lipases and esterases) and a p-nitroanilide substrate (to measure the activity of the proteinase VCP1). Additionally, information on parasitic growth (nematode egg parasitism) and saprotrophic growth (plant rhizosphere colonisation) was collected. Results showed that the production of extracellular enzymes was influenced by the type of medium (p < 0.05) in which P. chlamydosporia was grown. Enzyme activity differed with time (p < 0.05), and significant differences were found between isolates (p < 0.001) and the amounts of enzymes produced (p < 0.001). However, no significant relationships were found between enzyme activities and parasitic or saprotrophic growth using Kendall's coefficient of concordance or Spearman rank correlation coefficient. The results provided new information about enzyme production in P. chlamydosporia and suggested that the mechanisms which regulate the trophic switch in this fungus are complex and dependent on several factors.     

A study on the enzyme catalysed enantioselective hydrolysis of methyl 2-methyl-4-oxopentanoate,a precursor of chiral γ-butyrolactones

Porcine pancreas lipase (PPL) resolution of the α-methyl group of racemic methyl 2-methyl-4-oxopentanoate, a valuable synthetic precursor of fragrances and marine natural products, was enhanced by salt modulation of the enzymatic hydrolysis. For the enantioselective hydrolysis of the title ester, PPL was selected from a series of esterases and lipases, and its enantioselectivity was evaluated by changing the reaction medium parameters. The use of 1.6?mol L^–1 sodium sulfate in phosphate buffer (pH 7.2) improved the enantioselectivity allowing the formation of methyl (2R)-(+)-2-methyl-4-oxopentanoate and (2S)-(–)-2-methyl-4-oxopentanoic acid with an enantiomeric excess of >99% and 71%, respectively. The study showed that a modulation of PPL enantioselectivity could be achieved by using kosmotropic salts in the reaction media. The present method consists of a practical and low-cost option to improve enzymatic kinetic resolution reactions.     

Probing the substrate specificity for lipases. II. Kinetic and modeling studies on the molecular recognition of 2-arylpropionic esters by Candida rugosa and Rhizomucor miehei lipases

Racemic arylpropionic esters 1–3, precursors of therapeutically important non-steroidal antiinflammatory drugs, were subjected to hydrolyses in the presence of either Candida rugosa or Rhizomucor miehei crude lipases. The hydrolyses of 1 and 2 proved to be highly enantioselective, whereas 3 was not transformed at all. Both the substrate specificity and the enantioselectivity of these lipases were explained through a molecular modeling study involving docking experiments between 1–3 and the amino acids forming the enzymes active-sites, whose three-dimensional structures were obtained from X-ray crystallographic data, followed by extensive conformational analysis on their computer-generated complexes. The results of this study also account for the high enantioselective and good yielding hydrolysis of 3 (as the corresponding 2-chloroethyl ester) catalyzed by CRL pretreated with 2-propanol, recently reported in the literature, and lead to admit that such a treatment may operate very deep conformational changes on the amino acids of the enzyme active-site.     

Structure,mechanism, and enantioselectivity shifting of lipase LipK107 with a simple way

Because of the complex mechanisms of enzymatic reactions, no precise and simple method of understanding and controlling the chiral selectivity of enzymes has been developed. However, structure-based rational design is a powerful approach to engineering enzymes with desired catalytic activities. In this work, a simple, structure-based, large-scale in silico design and virtual screening strategy was developed and successfully applied to enzyme engineering. We first performed protein crystallization and X-ray diffraction to determine the structure of lipase LipK107, which is a novel family I.1 lipase displaying activity for both R and S isomers in chiral resolution reactions. The catalytic mechanism of family I.1, which includes LipK107, was ascertained first through comparisons of the sequences and structures of lipases from other families. The binding states of LipK107, including the energy and the conformation of complexes with the R and S enantiomers, have been evaluated by careful biocomputation to figure out the reason for the higher S selectivity. Based on this study, a simple strategy for manipulating the chiral selectivity by modulating a crucial distance in the enzyme–substrate complex and judging virtual mutations in silico is recommended. Then, a novel electrostatic interaction analysis protocol was used to design LipK107 mutants to validate our strategy. Both positive and negative mutations determined using this theoretical protocol have been implemented in wet experiments and were proved to produce the desired enantioselectivity, showing a 176% increase or 50% decrease in enantioselectivity as desired. Because of its accuracy and versatility, the strategy is promising for practical applications.     

Enzymatic synthesis of geraniol esters in a solvent-free system by lipases

Geraniol esters were synthesised by direct esterification catalysed by esterases and lipases (five enzymes were tested) in a solvent-free system at 37°C. The best conversions yields, about 85%, on geranyl butyrate and valerate obtained with esterase 30 000 from Mucor miehei. The effect of substrate molar ratio alcohol/acid variation was studied. A study of the water production was made in parallel during the esterification reaction.     

Enantioseparation of chiral pharmaceuticals by vancomycin‐bonded stationary phase and analysis of chiral recognition mechanism

The drug chirality is attracting increasing attention because of different biological activities, metabolic pathways, and toxicities of chiral enantiomers. The chiral separation has been a great challenge. Optimized high‐performance liquid chromatography (HPLC) methods based on vancomycin chiral stationary phase (CSP) were developed for the enantioseparation of propranolol, atenolol, metoprolol, venlafaxine, fluoxetine, and amlodipine. The retention and enantioseparation properties of these analytes were investigated in the variety of mobile phase additives, flow rate, and column temperature. As a result, the optimal chromatographic condition was achieved using methanol as a main mobile phase with triethylamine (TEA) and glacial acetic acid (HOAc) added as modifiers in a volume ratio of 0.01% at a flow rate of 0.3 mL/minute and at a column temperature of 5°C. The thermodynamic parameters (eg, ΔH, ΔΔH, and ΔΔS) from linear van 't Hoff plots revealed that the retention of investigated pharmaceuticals on vancomycin CSP was an exothermic process. The nonlinear behavior of lnk′ against 1/T for propranolol, atenolol, and metoprolol suggested the presence of multiple binding mechanisms for these analytes on CSP with variation of temperature. The simulated interaction processes between vancomycin and pharmaceutical enantiomers using molecular docking technique and binding energy calculations indicated that the calculated magnitudes of steady combination energy (ΔG) coincided with experimental elution order for most of these enantiomers.     

Biochemical Diversity of Carboxyl Esterases and Lipases from Lake Arreo (Spain): a Metagenomic Approach

The esterases and lipases from the α/β hydrolase superfamily exhibit an enormous sequence diversity, fold plasticity, and activities. Here, we present the comprehensive sequence and biochemical analyses of seven distinct esterases and lipases from the metagenome of Lake Arreo, an evaporite karstic lake in Spain (42°46′N, 2°59′W; altitude, 655 m). Together with oligonucleotide usage patterns and BLASTP analysis, our study of esterases/lipases mined from Lake Arreo suggests that its sediment contains moderately halophilic and cold-adapted proteobacteria containing DNA fragments of distantly related plasmids or chromosomal genomic islands of plasmid and phage origins. This metagenome encodes esterases/lipases with broad substrate profiles (tested over a set of 101 structurally diverse esters) and habitat-specific characteristics, as they exhibit maximal activity at alkaline pH (8.0 to 8.5) and temperature of 16 to 40°C, and they are stimulated (1.5 to 2.2 times) by chloride ions (0.1 to 1.2 M), reflecting an adaptation to environmental conditions. Our work provides further insights into the potential significance of the Lake Arreo esterases/lipases for biotechnology processes (i.e., production of enantiomers and sugar esters), because these enzymes are salt tolerant and are active at low temperatures and against a broad range of substrates. As an example, the ability of a single protein to hydrolyze triacylglycerols, (non)halogenated alkyl and aryl esters, cinnamoyl and carbohydrate esters, lactones, and chiral epoxides to a similar extent was demonstrated.