A simple activity staining protocol for lipases and esterases

A simple activity staining protocol for rapid detection and differentiation of lipases and esterases was developed based on pH drop due to fatty acids released following lipolysis. Though the detection of lipolysis as a function of drop in pH is not new, the present method has been made more sensitive by the judicious selection of the initial pH of the chromogenic substrate, which has been set near the end point of the dye so that even a slight drop in pH results in immediate color change. In the present case, the dye phenol red was taken, which has the end point at pH 7.3–7.4 where the color is pink. A slight drop due to fatty acid release results in yellow coloration. The assay has high reproducibility and can detect as low as 0.5 p-NPP enzyme units within 15 min. In addition, this method can be used for various lipidic substrates such as oils and tributyrin, making it suitable for both lipases and esterases.     

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.     

Resolution of 4-amino-cyclopentanecarboxylic acid methyl esters using hydrolytic enzymes.

A number of esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3) of microbial and mammalian origin were screened for the ability to resolve racemic 4-amino-cyclopentanecarboxylic acid methyl ester derivatives as potential intermediates in the production of carbocyclic nucleosides. Surprisingly, functionalization of the remote amino group had a profound effect on both the rate and enantioselectivity of hydrolysis of the methyl ester. 4-(Benzoylamino)-2-cyclopentenecarboxylic acid, methyl ester (V) with pig liver esterase gave the highest enantioselectivity. The residual ester, which was of the correct absolute stereochemistry [(+) 1S, 4R] for carbocyclic nucleoside synthesis, could be obtained in high optical purity. Optimization of pH, solvent type, and concentration improved the enantioselectivity of the process by a further twofold.     

Identification of novel esterases for the synthesis of sterically demanding chiral alcohols by sequence-structure guided genome mining

Six esterases isolated from sequence and structure-guided genome mining approaches were evaluated for the kinetic resolution of secondary and tertiary alcohols that find application in the fine chemical and pharmaceutical industries. Activity and enantioselectivity with E-values of up to 24 were determined towards a range of sterically demanding tertiary alcohol esters. Excellent enantioselectivity (E > 100) was also achieved in the hydrolysis of a less challenging secondary alcohol ester, menthyl acetate. These results highlight that these approaches can be used for the identification of novel esterases applicable to the preparation of commercially desirable alcohols.     

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.     

A high-throughput amenable colorimetric assay for enantioselective screening of nitrilase-producing microorganisms using pH sensitive indicators

Based on the color change of an indicator due to the release of hydrogen ion from a nitrilase-catalyzed reaction, a rapid colorimetric method was established for the enantioselective screening of nitrilase-producing microorganisms. The formation of acids due to the nitrilase-mediated hydrolysis of nitriles causes a drop in the pH, which in turn results in a change of color of the solution (containing indicator) that can be observed visually. The buffer (0.01 M phosphate, pH 7.2) and indicator (Bromothymol blue, 0.01%) were selected in such a way that both have the same affinity for the released protons. The enantioselectivity of nitrilases was estimated by comparing the hydrolysis of (R)-mandelonitrile with that of racemate under the same conditions. The method was used to screen a library of nitrilase-producing microorganisms, isolated in the authors' laboratory for their ability to enantioselectively hydrolyze mandelonitrile to mandelic acid, an important chiral building block.     

High-throughput screening of activity and enantioselectivity of esterases

A procedure for the high-throughput screening of esterases is described. This includes enzyme expression in microtiter plates and the measurement of activity and enantioselectivity (E) of the esterase variants using acetates of secondary alcohols as model substrates. Acetic acid released is converted in an enzyme cascade leading to the stoichiometric formation of NADH, which is quantified in a spectrophotometer. The method allows screening of several thousand mutants per day and has already been successfully applied to identify an esterase mutant with an E>100 toward an important building block for organic synthesis. This protocol can also be used for lipases and possibly other hydrolases that are expressed in soluble form in conventional Escherichia coli strains. This protocol can be completed in 3-4 days.     

Methods to increase enantioselectivity of lipases and esterases

Lipases and esterases are frequently used in the synthesis of optically pure compounds; however, natural enzymes do not always show sufficiently high enantioselectivity. Variation of the structure of the substrates, modification of the reaction system or protein engineering (e.g. the expression of pure enzymes, rational design or directed evolution) are strategies that can be employed to improve the distinction between two enantiomers or enantiotopic groups.     

Screening and characterization of a novel esterase from a metagenomic library

Metagenomes from various environmental soils were screened using alpha-naphthyl acetate and Fast Blue RR for a novel ester-hydrolyzing enzyme on Escherichia coli. Stepwise fragmentations and subcloning of the initial insert DNA (30-40 kb) using restriction enzymes selected to exclude already known esterases with subsequent screenings resulted in a positive clone with a 2.5-kb DNA fragment. The cloned sequence included an open reading frame consisting of 1089 bp, designated as est25, encoding a protein of 363 amino acids with a molecular mass of about 38.3 kDa. Amino acid sequence analysis revealed only moderate identity (< or = 48%) to the known esterases/lipases in the databases containing the conserved sequence motifs of esterases/lipases, such as HGGG (residues 124-127), GxSxG (residues 199-203), and the putative catalytic triad composed of Ser201, Asp303, and His333. Est25 was functionally overexpressed in a soluble form in E. coli with optimal activity at pH 7.0 and 25 degrees C. The purified Est25 exhibited hydrolyzing activity toward p-nitrophenyl (NP)-fatty acyl esters with short-length acyl chains (< or = C6) with the highest activity toward p-NP-acetate (Km=1.0 mM and Vmax = 63.7 U/mg), but not with chain lengths > or = C8, demonstrating that Est25 is an esterase originated most likely from a mesophilic microorganism in soils. Est25 efficiently hydrolyzed (R,S)-ketoprofen ethyl ester with Km of 16.4 mM and Vmax of 59.1 U/mg with slight enantioselectivity toward (R)-ketoprofen ethyl ester. This study demonstrates that functional screening combined with the sequential uses of restriction enzymes to exclude already known enzymes is a useful approach for isolating novel enzymes from a metagenome.     

Chaperone-like activities of alpha-synuclein: alpha-synuclein assists enzyme activities of esterases

Alpha-synuclein, a major constituent of Lewy bodies (LBs), has been implicated to play a critical role in the pathogenesis of Parkinson's disease (PD), although the physiological function of alpha-synuclein has not yet been known. Here we have shown that alpha-synuclein, which has no well-defined secondary or tertiary structure, can protect the enzyme activity of microbial esterases against stress conditions such as heat, pH, and organic solvents. In particular, the flexibility of alpha-synuclein and its C-terminal region seems to be important for complex formation, but the structural integrity of the C-terminal region may not be required for stabilization of enzyme activity. In addition, atomic force microscopy (AFM) and in vivo enzyme assays showed highly specific interactions of esterases with alpha-synuclein. Our results indicate that alpha-synuclein not only protects the enzyme activity of microbial esterases in vitro, but also can stabilize the active conformation of microbial esterases in vivo.     

Esterases in mycobacteria

It was found that a submerged culture ofMycobacterium phlei degrades simple esters (ethylacetate and ethylbutyrate) as well as synthetic lipids (triacetine and tributyrine). The effect of pH on the rate of degradation of tributyrine was investigated and the maximum activity of esterases found within a wide range of pH. The activity of esterases was followed during growth of a submerged culture ofMycobacterium phlei. Esterases were not released into the cultivation medium during growth or even during the early stationary phase. Only a low steady activity of esterases could be demonstrated in a filtrate of the cultivation liquid. The total activity of esterases reached its maximum after a 6–11 day incubation. The specific activity of esterases reached a maximum on the 6th day of incubation; its value decreased to about one half and did not change substantially on prolonged incubation. Changes in the specific activity of esterases were found to be time-related with changes of pH and a decrease of the specific activity was associated with a release of macromolecular compounds into the incubation medium. Esterases as well as other macromolecular compounds were isolated from the filtrate of the cultivation medium ofMycobacterium phlei. The isolated preparation contained 60–72% total activity of esterases present in the filtrate of the cultivation liquid.     

Isolation of Two Acetyl Esterases from Extracts of Bacillus subtilis

Acrylamide gel electrophoresis of crude cellular extracts of Bacillus subtilis revealed the presence of two acetyl esterases. Esterase A, the slower migrating enzyme, was found to be present in both vegetative and sporulating cells, whereas esterase B activity was more abundant after exponential growth ceased. Both esterases were present in the supernatant fraction of lysed spheroplasts and in a disrupted spore preparation. Of four pleiotropic asporogenous mutants tested, three exhibited decreased esterase B activity. Esterases A and B were partially purified by differential precipitation and co-chromatographed on diethylaminoethyl (DEAE)-cellulose (pH 7.5) and DEAE-Sephadex (pH 8.5). By employing gel filtration chromatography, the two esterases were separated, and molecular weights of 160,000 and 51,000 were estimated for esterases A and B, respectively. Esterase A was further purified to electrophoretic homogeneity by differential heating and preparative starch block electrophoresis. Sodium dodecyl sulfate-acrylamide gel electrophoresis of purified esterase A yielded a single protein band with a molecular weight of 31,000. The pI values of esterases A and B were determined to be 6.4 and 5.4, respectively.     

In vitro hydrolysis of RR,SS-threo-methylphenidate by blood esterases--differential and enantioselective interspecies variability

Enantioselective in vitro hydrolysis of methylphenidate (MPH) by the blood esterases of seven mammalian species is reported. The species included rats, rabbits, dogs, cattle, horses, monkeys, and humans. In vitro incubations up to 8 h were carried out in plasma, red blood cells, and whole blood of the various species. Enantioselective differences were evident among the different species on comparison of the data obtained from the three biological fluids. The esterases present in plasma appeared to show greater activity in the hydrolysis of MPH in all species where comparison with the other two biofluids was possible. Only in the case of humans did esterases present in plasma and red blood cells demonstrate opposite enantioselectivity in the hydrolysis of MPH. Thus after 8 h incubation, the RR-MPH/SS-MPH ratios in plasma and red blood cells were 0.31 and 1.16, respectively.     

Cloning and characterization of thermostable esterase from <Emphasis Type="Italic">Archaeoglobus fulgidus</Emphasis>

Thermostable esterase gene was cloned (Est-AF) from extremophilic microorganisms, Archaeoglobus fulgidus DSM 4304. The protein analysis result showed that Est-AF is monomer with total 247 amino acids and molecular weight of estimated 27.5 kDa. It also showed repeating units G-X-S-X-G (GHSLG) (residues 86 approximately 90) which is reported as active site of known esterases, and the putative catalytic triad composed of Ser88, Asp198 and His226. The esterase activity test with various acyl chain length of rho-nitrophenol resulted that Est-AF showed highest specific activity with rho-nitrophenylbutyrate (pNPC4) and rapidly decrease with rho-nitrophenyl ester contain more than 8 carbon chain. These results represent that cloned enzyme is verified as a carboxylesterase but not a lipase because esterase activity is decreased with rho-nitrophenyl ester contains more than 8 carbon chains but lipase activity does not affected with carbon chain length. Optimum temperature of esterase reaction with rho-nitrophenylbutyrate (pNPC4) was 80 degrees C. When ketoprofen ethyl ester was used as a substrate, activity of Est-AF showed the highest value at 70 degrees C, and 10% of activity still remains after 3 h of incubation at 90 degrees C. This result represents Est-AF has high thermostability with comparison of other esterases that have been reported. However, Est-AF showed low enantioselectivity with ketoprofen ethyl ester. Optimum pH of Est-AF is between pH 7.0 and pH 8.0. Km value of ketoprofen ethyl ester is 1.6 mM and, Vmax is 1.7 micromole/mg protein/min. Est-AF showed similar substrate affinity but slower reaction with ketoprofen ethyl ester compare with esterase from mesophilic strain P. fluorescens.     

Purification and characterization of two thermostable acetyl xylan esterases from Thermoanaerobacterium sp. strain JW/SL-YS485.

Two acetyl esterases (EC 3.1.1.6) were purified to gel electrophoretic homogeneity from Thermoanaerobacterium sp. strain JW/SL-YS485, an anaerobic, thermophilic endospore former which is able to utilize various substituted xylans for growth. Both enzymes released acetic acid from chemically acetylated larch xylan. Acetyl xylan esterases I and II had molecular masses of 195 and 106 kDa, respectively, with subunits of 32 kDa (esterase I) and 26 kDa (esterase II). The isoelectric points were 4.2 and 4.3, respectively. As determined by a 2-min assay with 4-methylumbelliferyl acetate as the substrate, the optimal activity of acetyl xylan esterases I and II occurred at pH 7.0 and 80 degrees C and at pH 7.5 and 84 degrees C, respectively. Km values of 0.45 and 0.52 mM 4-methylumbelliferyl acetate were observed for acetyl xylan esterases I and II, respectively. At pH 7.0, the temperatures for the 1-h half-lives for acetyl xylan esterases I and II were 75 degrees and slightly above 100 degrees C, respectively.     

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 (ΔΔG_docking) was significantly (correlation coefficient = 0.72, P < 0.05) correlated with the activation free energy difference (ΔΔG^≠) 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 ΔΔG^≠ 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. Biotechnol. Bioeng. 2010. 105: 687–696. © 2009 Wiley Periodicals, Inc.     

Purification and properties of three esterases from Brevibacterium sp. R312

C. LAMBRECHTS, J. ESCUDERO AND P. GALZY. 1995. The esterases of Brevibacterium sp. R312 were found to have an intracellular location. Electrophoresis of lysed cell supernatant fluids revealed seven bands of esterase activity in the presence of α-naphthyl acetate. Eight esterases were separated by anion exchange chromatography. The three main esterases (esterase 4b, 2 and 4a) of Brevibacterium sp. R312 were purified. The molar masses, the pH optima, the temperature optima and heat stabilities were determined. Esterase 2 differed from the two others in sensitivity to inhibitors. Esterase 4b differed from esterases 2 and 4a in its substrate specificity. This enzyme hydrolyses aliphatic and nitrophenyl esters. The spectrum of activity of the two other esterases is narrower. They hydrolysed only naphthyl esters and, in the case of esterase 2, tributyrate and ethyl butyrate.     

A biochemical study of non-specific esterases from plant cells, employing the histochemical substrate, naphthol AS-D acetate

Synopsis The reagents routinely employed in the histochemical detection of nonspecific esterases, namely naphthol AS-D acetate and Fast Red Violet LB salt, have been used to study these enzymes biochemically with spectrophotometric procedures. A range of parameters that affect the coupling of the hydrolyzed substrate and diazonium salt were examined and their relevance to future histochemical procedures is noted.TheK _m of broad-bean root-tip esterases was estimated to be approximately 0.07 mM, but other kinetic data suggest that the true value is lower. From an analysis of the kinetics of the hydrolytic reaction it appears that they are of a mixed nature over the time course and substrate concentrations used.The effect of pH on root tip esterase activity has been examined and the recorded optimum of 5.5 is similar to that reported by other workers for plant cells. Non-enzymic hydrolysis of the substrate at alkaline pH levels prevented measurement of enzyme activity beyond pH 7.2.Magnesium ions at a final concentration of 5 mM are important in retaining esterases in their natured state during enzyme extraction, although the addition of increasing amounts of the ion to the assay system caused a corresponding increase in esterase inhibition.     

Synthesis and catalytic activity of polypeptides of regular structure containing polyfunctional amino acids

Synthesis and study of catalytic properties of a series of regular polypeptide which contain residues of polyfunctional amino acids forming the active centre of esterases are described viz. (Ala-Tyr-Glu)n, (His-Ser-Glu)n, (Glu-His-Glu)n, (His-Glu)n, (Ser-His-Glu)n, His-(Tyr-Glu)n. Possibility of constructing catalytically active model polypeptides which can substitute some hydrolytic enzymes is assessed. Monomers for polycondensation were been synthesized by carbodiimide method in solution, and polypeptides were obtained via 2,4,5-trichlorphenylates. Gel-filtration was used for fractionation of and molecular mass-determination of polypeptides. Catalytic properties of the synthetic polypeptides were studied in hydrolysis of p-NFA, Z-L-Ala-ONp, and Z-D-Ala-ONp. It was revealed that polypeptides (Ala-Tyr-Glu)n and (His-Ser-Glu)n possess, in hydrolysis of Z-L-Ala-ONp- and Z-D-Ala-ONp some enantioselectivity with the value of KD/KL 1.3 and 1.17, resp. The upper and lower limits of enantioselectivity as dependent of the molecular mass of the corresponding polypeptides have been determined.