P-nitrophenyl Esters Of Fatty Acids Research Articles

Studies on the catalytic behavior of a membrane-bound lipolytic enzyme from the microalgae Nannochloropsis oceanica CCMP1779

The catalytic behavior of a membrane-bound lipolytic enzyme (MBL-Enzyme) from the microalgae Nannochloropsis oceanica CCMP1779 was investigated. The biocatalyst showed maximum activity at 50 °C and pH 7.0, and was stable at pH 7.0 and temperatures from 40 to 60 °C. Half-lives at 60 °C, 70 °C and 80 °C were found 866.38, 150.67 and 85.57 min respectively. Thermal deactivation energy was 68.87 kJ mol−1. The enzyme’s enthalpy (ΔΗ*), entropy (ΔS*) and Gibb’s free energy (ΔG*) were in the range of 65.86–66.27 kJ mol−1, 132.38–140.64 J mol−1 K−1 and 107.80–115.81 kJ mol−1, respectively. Among p-nitrophenyl esters of fatty acids tested, MBL-Enzyme exhibited the highest hydrolytic activity against p-nitrophenyl palmitate (pNPP). The Km and Vmax values were found 0.051 mM and of 0.054 mmole pNP mg protein−1 min−1, respectively with pNPP as substrate. The presence of Mn2+ increased lipolytic activity by 68.25%, while Fe3+ and Cu2+ ions had the strongest inhibitory effect. MBL-Enzyme was stable in the presence of water miscible (66% of the initial activity in ethanol) and water immiscible (71% of the initial activity in n-octane) solvents. Myristic acid was found to be the most efficient acyl donor in esterification reactions with ethanol. Methanol was the best acyl acceptor among the primary alcohols tested.

Production and characterization of a tributyrin esterase from Lactobacillus plantarum suitable for cheese lipolysis

Lactobacillus plantarum is a lactic acid bacterium that can be found during cheese ripening. Lipolysis of milk triacylglycerols to free fatty acids during cheese ripening has fundamental consequences on cheese flavor. In the present study, the gene lp_1760, encoding a putative esterase or lipase, was cloned and expressed in Escherichia coli BL21 (DE3) and the overproduced Lp_1760 protein was biochemically characterized. Lp_1760 hydrolyzed p-nitrophenyl esters of fatty acids from C2 to C16, with a preference for p-nitrophenyl butyrate. On triglycerides, Lp_1760 showed higher activity on tributyrin than on triacetin. Although optimal conditions for activity were 45°C and pH 7, Lp_1760 retains activity under conditions commonly found during cheese making and ripening. The Lp_1760 showed more than 50% activity at 5°C and exhibited thermal stability at high temperatures. Enzymatic activity was strongly inhibited by sodium dodecyl sulfate and phenylmethylsulfonyl fluoride. The Lp_1760 tributyrin esterase showed high activity in the presence of NaCl, lactic acid, and calcium chloride. The results suggest that Lp_1760 might be a useful tributyrin esterase to be used in cheese manufacturing.

Molecular cloning of a novel bioH gene from an environmental metagenome encoding a carboxylesterase with exceptional tolerance to organic solvents

BackgroundBioH is one of the key enzymes to produce the precursor pimeloyl-ACP to initiate biotin biosynthesis de novo in bacteria. To date, very few bioH genes have been characterized. In this study, we cloned and identified a novel bioH gene, bioHx, from an environmental metagenome by a functional metagenomic approach. The bioHx gene, encoding an enzyme that is capable of hydrolysis of p-nitrophenyl esters of fatty acids, was expressed in Escherichia coli BL21 using the pET expression system. The biochemical property of the purified BioHx protein was also investigated.ResultsScreening of an unamplified metagenomic library with a tributyrin-containing medium led to the isolation of a clone exhibiting lipolytic activity. This clone carried a 4,570-bp DNA fragment encoding for six genes, designated bioF, bioHx, fabG, bioC, orf5 and sdh, four of which were implicated in the de novo biotin biosynthesis. The bioHx gene encodes a protein of 259 aa with a calculated molecular mass of 28.60 kDa, displaying 24-39% amino acid sequence identity to a few characterized bacterial BioH enzymes. It contains a pentapeptide motif (Gly76-Trp77-Ser78-Met79-Gly80) and a catalytic triad (Ser78-His230-Asp202), both of which are characteristic for lipolytic enzymes. BioHx was expressed as a recombinant protein and characterized. The purified BioHx protein displayed carboxylesterase activity, and it was most active on p-nitrophenyl esters of fatty acids substrate with a short acyl chain (C4). Comparing BioHx with other known BioH proteins revealed interesting diversity in their sensitivity to ionic and nonionic detergents and organic solvents, and BioHx exhibited exceptional resistance to organic solvents, being the most tolerant one amongst all known BioH enzymes. This ascribed BioHx as a novel carboxylesterase with a strong potential in industrial applications.ConclusionsThis study constituted the first investigation of a novel bioHx gene in a biotin biosynthetic gene cluster cloned from an environmental metagenome. The bioHx gene was successfully cloned, expressed and characterized. The results demonstrated that BioHx is a novel carboxylesterase, displaying distinct biochemical properties with strong application potential in industry. Our results also provided the evidence for the effectiveness of functional metagenomic approach for identifying novel bioH genes from complex ecosystem.

Identification of novel lipolytic genes and gene families by screening of metagenomic libraries derived from soil samples of the German Biodiversity Exploratories

Microbial metagenomes derived from soils are rich sources for the discovery of novel genes and biocatalysts. Fourteen environmental plasmid and seven fosmid libraries obtained from 10 German forest soils (A horizons) and six grassland soils (A and B horizons) were screened for genes conferring lipolytic activity. The libraries comprised approximately 29.3 Gb of cloned soil DNA. Partial activity-based screening of the constructed libraries resulted in the identification of 37 unique lipolytic clones. The amino acid sequences of the 37 corresponding lipolytic gene products shared 29-90% identity to other lipolytic enzymes, which were mainly uncharacterized or derived from uncultured microorganisms. Multiple sequence alignments and phylogenetic tree analysis revealed that 35 of the predicted proteins were new members of known families of lipolytic enzymes. The remaining two gene products represent two putatively new families. In addition, sequence analysis indicated that two genes encode true lipases, whereas the other genes encode esterases. The determination of substrate specificity and chain-length selectivity using different triacylglycerides and p-nitrophenyl esters of fatty acids as substrates supported the classification of the esterases.

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.

Purification and Characterization of an Organic Solvent-Tolerant Lipase from Pseudomonas aeruginosa CS-2

An extracellular lipase secreted by Pseudomonas aeruginosa CS-2 was purified to homogeneity about 25.5-fold with an overall yield of 45.5%. The molecular mass of the lipase was estimated to be 33.9 kDa by SDS-PAGE and 36 kDa by gel filtration. The optimum temperature and pH were 50 degrees C and 8.0. The lipase was found to be stable at pH 4-10 and below 50 degrees C. Its hydrolytic activity was highest against p-nitrophenyl palmitate (p-NPP) among p-nitrophenyl esters of fatty acids with various chain lengths. The lipase was activated in the presence of Ca(2+), while it was inactivated by other metal ions more or less. EDTA significantly reduced the lipase activity, indicating the lipase was a metalloenzyme. Gum Arabic and polyvinyl alcohol 124 enhanced lipase activity but Tween-20, Tween-80, and hexadecyltrimethyl ammonium bromide strongly inhibited the lipase. It exhibited stability in some organic solvents. The lipase was activated in the presence of acetonitrile. Conversely, it was drastically inactivated by methanol and ethanol.

A high throughput profiling method for cutinolytic esterases

A procedure for identifying and profiling cutinolytic esterases was developed by combining traditional plate screen assays with an automated robotic system. In the first phase, the micro-organisms were screened on agar plates with cutin or the model substrate polycaprolactone as the sole carbon sources. In the second phase, p-nitrophenyl esters of fatty acids were used as the substrates in an automated activity assay of liquid culture media. The variables used were pH and the carbon chain length of the fatty acid moiety of the p-nitrophenyl substrate. Finally, 3H-labelled cutin was used as a specific substrate to verify the positive hits and to validate the screening procedure. With pH as the variable in the automatic screen, esterase production of cutinase positive strains typically proceeded in two stages: first an esterase with neutral activity optimum was produced, after which a strong esterolytic response in the alkaline range was detected. With carbon chain length of the fatty acid as the variable best correlation with cutinase production was obtained with strains showing a high ratio of activities towards p-nitrophenyl-butyrate and p-nitrophenyl-palmitate.

Biochemical properties and potential applications of an organic solvent-tolerant lipase isolated from Serratia marcescens ECU1010

An extracellular lipase was purified to homogeneity with a purification factor of 5.5-fold from a bacterial strain Serratia marcescens ECU1010. The purified lipase is a dimer with two homologous subunits, of which the molecular mass is 65 kDa, and the p I is 4.2. The pH and temperature optima were shown to be pH 8.0 and 45 °C, respectively. Among p-nitrophenyl esters of fatty acids with varied chain length, the lipase showed the maximum activity on p-nitrophenyl myristate (C 14). The lipase was activated by some surfactants such as Gum Arabic, polyvinyl alcohol (PVA) and Pg350me, but not by Ca 2+. The enzyme displayed pretty high stability in many water miscible and immiscible solvents. This is a unique property of the enzyme which makes it extremely suitable for chemo-enzymatic applications in non-aqueous phase organic synthesis including enantiomeric resolution. Several typical chiral compounds were tested for kinetic resolution with this lipase, consequently giving excellent enantioselectivities ( E = 83 ∼>100) for glycidyl butyrate (GB), 4-hydroxy-3-methyl-2-(2-propenyl)-2-cyclopenten-1-one acetate (HMPCA), naproxen methyl ester (NME) and trans-3-(4′-methoxyphenyl) glycidic acid methyl ester (MPGM).

Screening and its potential application of lipolytic activity from a marine environment: characterization of a novel esterase from Yarrowia lipolytica CL180

To develop an enantioselective lipase/esterase hydrolyzing racemic ofloxacin ester to levofloxacin, samples were collected from a variety of marine environments such as cold sea, hydrothermal vent area, sediment, tidal flat area, arctic sea, marine organisms, and so on. Microorganisms were isolated by plating on an enrichment medium with simultaneous detection of lipolytic activities and screened for the hydrolysis of ofloxacin ester. Three candidates among isolates were selected, and one of them, identified as Yarrowia lipolytica CL180, hydrolyzed preferentially S-enantiomer of racemic ofloxacin ester. The lipase/esterase gene (yli180) was cloned by screening a genomic library. The sequence analysis revealed an open reading frame consisting of 1,431 bp that encoded a protein of 476 amino acids with a molecular mass of 53 kDa. The yli180 gene was expressed in Escherichia coli and purified to homogeneity. The optimum activity of the recombinant protein (rYli180) occurred at pH 7.5 and 35 degrees C, respectively. rYli180 preferentially hydrolyzed p-nitrophenyl esters of fatty acids with short chain lengths of < or =10 carbon atoms. This study represents a novel esterase of type B1 carboxylesterase/lipase family from a marine isolate, showing a potential usage as a biocatalyst because of enantioselectivity toward racemic ofloxacin ester.

Metagenomic approach for the isolation of a novel low-temperature-active lipase from uncultured bacteria of marine sediment

A novel lipase was isolated from a metagenomic library of Baltic Sea sediment bacteria. Prokaryotic DNA was extracted and cloned into a copy control fosmid vector (pCC1FOS) generating a library of >7000 clones with inserts of 24-39 kb. Screening for clones expressing lipolytic activity based on the hydrolysis of tributyrin and p-nitrophenyl esters, identified 1% of the fosmids as positive. An insert of 29 kb was fragmented and subcloned. Subclones with lipolytic activity were sequenced and an open reading frame of 978 bp encoding a 35.4-kDa putative lipase/esterase h1Lip1 (DQ118648) with 54% amino acid similarity to a Pseudomonas putida esterase (BAD07370) was identified. Conserved regions, including the putative active site, GDSAG, a catalytic triad (Ser148, Glu242 and His272) and a HGG motif, were identified. The h1Lip1 lipase was over expressed, (pGEX-6P-3 vector), purified and shown to hydrolyse p-nitrophenyl esters of fatty acids with chain lengths up to C14. Hydrolysis of the triglyceride derivative 1,2-di-O-lauryl-rac-glycero-3-glutaric acid 6'-methylresorufin ester (DGGR) confirmed that h1Lip1 was a lipase. The apparent optimal temperature for h1Lip1, by hydrolysis of p-nitrophenyl butyrate, was 35 degrees C. Thermal stability analysis showed that h1Lip1 was unstable at 25 degrees C and inactivated at 40 degrees C with t1/2 <5 min.

Molecular Basis of Formaldehyde Detoxification

The Escherichia coli genes frmB (yaiM) and yeiG encode two uncharacterized proteins that share 54% sequence identity and contain a serine esterase motif. We demonstrated that purified FrmB and YeiG have high carboxylesterase activity against the model substrates, p-nitrophenyl esters of fatty acids (C2-C6) and alpha-naphthyl acetate. However, both proteins had the highest hydrolytic activity toward S-formylglutathione, an intermediate of the glutathione-dependent pathway of formaldehyde detoxification. With this substrate, both proteins had similar affinity (Km = 0.41-0.43 mM), but FrmB was almost 5 times more active. Alanine replacement mutagenesis of YeiG demonstrated that Ser145, Asp233, and His256 are absolutely required for activity, indicating that these residues represent a serine hydrolase catalytic triad in this protein and in other S-formylglutathione hydrolases. This was confirmed by inspecting the crystal structure of the Saccharomyces cerevisiae S-formylglutathione hydrolase YJG8 (Protein Data Bank code 1pv1), which has 45% sequence identity to YeiG. The structure revealed a canonical alpha/beta-hydrolase fold and a classical serine hydrolase catalytic triad (Ser161, His276, Asp241). In E. coli cells, the expression of frmB was stimulated 45-75 times by the addition of formaldehyde to the growth medium, whereas YeiG was found to be a constitutive enzyme. The simultaneous deletion of both frmB and yeiG genes was required to increase the sensitivity of the growth of E. coli cells to formaldehyde, suggesting that both FrmB and YeiG contribute to the detoxification of formaldehyde. Thus, FrmB and YeiG are S-formylglutathione hydrolases with a Ser-His-Asp catalytic triad involved in the detoxification of formaldehyde in E. coli.

Characterization of an extracellular medium-chain-length poly(3-hydroxyalkanoate) depolymerase from Streptomyces sp. KJ-72.

A bacterial strain capable of degrading medium-chain-length polyhydroxyalkanoates (MCL-PHAs) was isolated from a soil sample. This organism, which was identified as Streptomyces sp. KJ-72, secreted MCL-PHA depolymerase into the culture fluid only when it was cultivated on MCL-PHAs. The extracellular MCL-PHA depolymerase of the organism was purified to electrophoretic homogeneity by ion exchange column chromatography and gel filtration. The enzyme consisted of a monomeric subunit having a molecular mass of 27.1 kDa and isoelectric point of 4.7. The maximum activity was observed at pH 8.7 and 50 degrees C. The enzyme was sensitive to N-bromosuccinimide and acetic anhydride, indicating the presence of tryptophan and lysine residues in the catalytic domain. The enzyme was able to hydrolyze various chain-length p-nitrophenyl esters of fatty acids and polycaprolactone as well as various types of MCL-PHAs. However, lipase activity of the enzyme was not detected. The main hydrolysis product of poly(3-hydroxyheptanoate) was identified to be the dimer of 3-hydroxyheptanoate.

A carboxylesterase from the hyperthermophilic archaeon Sulfolobussolfataricus: cloning of the gene, characterization of the protein

A genomic library of the hyperthermophilic archaeon Sulfolobussolfataricus strain MT4 was constructed in Escherichiacoli using a cloning vector not designed for heterologous gene expression. One positive clone exhibiting acquired thermophilic acetylesterase activity was directly detected by an insitu plate assay using a colony staining procedure with the chromogenic substrate β-naphthyl acetate. The plasmid isolated from the clone contained a 3.3 kb genomic fragment from S.solfataricus and a full-length esterase coding sequence could be identified. Expression of the active thermostable esterase in E.coli was independent of isopropyl-β-d-thiogalactopyranoside and of the kind of vector, suggesting that the archaeal esterase gene was controlled by fortuitous bacterial-like sequences present in its own 5′ flanking region, not by the bacterial lac promoter or other serendipitous vector-located sequences. The protein, partially purified by thermoprecipitation of the host proteins at high temperature and gel exclusion chromatography, showed a homo-tetrameric structure with a subunit of molecular mass of 32 kDa which was in perfect agreement with that deduced from the cloned gene. The same protein was revealed in S.solfataricus cell extracts, thus demonstrating its functional occurrence invivo under the cell culture conditions tested. The recombinant enzyme exhibited high thermal activity and thermostability with optimal activity between pH 6.5 and 7.0. The hydrolysis of p-nitrophenyl esters of fatty acids (from C2 to C8) allowed the enzyme to be classified as a short length acyl esterase.

Purification and characterization of extracellular medium-chain-length polyhydroxyalkanoate depolymerase from Pseudomonas sp. RY-1

A novel bacterial strain capable of growing in a medium containing a medium-chain-length polyhydroxyalkanoate (MCL-PHA) as the sole carbon source was isolated from a soil sample. The isolate, which was identified as Pseudomonas sp. RY-1, secreted MCL-PHA depolymerase into the culture fluid only when it was cultivated in a medium containing a MCL-PHA, such as polyhydroxyoctanoate (PHO) or polyhydroxynonanoate (PHN). The extracellular MCL-PHA depolymerase of this organism was purified to electrophoretic homogeneity. The enzyme was a tetramer with identical subunits and a total molecular mass of 115 kDa. The isoelectric point of this enzyme was estimated to be 5.9 by isoelectric focusing. The maximal activity was observed at pH 8.5 and 35°C. The enzyme was insensitive to phenylmethylsulfonyl fluoride and dithiothreitol, unlike other short-chain-length (SCL) PHA depolymerases. The K m values for PHO and PHN were 0.86 and 1.47 mg/ml, respectively. The enzyme could not hydrolyze SCL-PHAs and p-nitrophenyl esters of fatty acids.

Identification of the gene encoding esterase, a homolog of hormone-sensitive lipase, from an oil-degrading bacterium, strain HD-1.

The gene encoding an esterase (HDE) was cloned from an oil-degrading bacterium, strain HD-1. HDE is a member of the hormone-sensitive lipase family and composed of 317 amino acid residues with a molecular weight of 33,633. The HDE-encoding gene was expressed in Escherichia coli, and the recombinant protein was purified and characterized. Amino acid sequence analysis indicated that the methionine residue was removed from its NH(2)-terminus. The good agreement of the molecular weights estimated by SDS-PAGE (35,000) and gel filtration (38,000) suggests that it acts in a monomeric form. HDE showed hydrolytic activity towards p-nitrophenyl esters of fatty acids with an acyl chain length of 2 to 14 and tributyrin, whereas it showed little hydrolytic activity towards p-nitrophenyl oleate (C(18)), tricaprylin and triolein. Determination of the kinetic parameters for the hydrolyses of the p-nitrophenyl substrates from C(2) to C(14) indicated that HDE shows a relatively broad substrate specificity. However, comparison of the k(cat)/K(m) values indicated that the C(10)-C(14) substrates are the most preferred ones. Such a preference for substrates with long acyl chains may be a characteristic of HDE.

An esterase from Escherichia coli with a sequence similarity to hormone-sensitive lipase.

An esterase from Escherichia coli that is a member of the hormone-sensitive lipase (HSL) family was overproduced, purified and characterized. It is encoded by the ybaC gene and composed of 319 amino acid residues with an Mr of 36038. The enzymic activity was determined by using various p-nitrophenyl esters of fatty acids as a substrate at 25 degreesC and pH 7.1. The enzyme showed hydrolytic activity towards substrates with an acyl chain length of less than 8, whereas it showed little hydrolytic activity towards those with an acyl chain length of more than 10. In addition, it showed little hydrolytic activity towards trioleoylglycerol and cholesterol oleate. Determination of the kinetic parameters for the hydrolyses of the substrates from C2 to C8 indicates that C4 and C5 substrates are the most preferred. Close agreement between the Mr determined by SDS/PAGE (37000) and column chromatography (38000) suggests that the enzyme exists in a monomeric form. It is an acidic protein with a pI value of 4.1. The far-UV CD spectrum suggests that its helical content is 26.1%. Comparison of the amino acid sequence of this enzyme with those involved in the HSL family allows us to propose that Ser165, Asp262 and His292 constitute the catalytic triad of E. coli esterase.

A cold-adapted lipase of an Alaskan psychrotroph, Pseudomonas sp. strain B11-1: gene cloning and enzyme purification and characterization.

A psychrotrophic bacterium producing a cold-adapted lipase upon growth at low temperatures was isolated from Alaskan soil and identified as a Pseudomonas strain. The lipase gene (lipP) was cloned from the strain and sequenced. The amino acid sequence deduced from the nucleotide sequence of the gene (924 bp) corresponded to a protein of 308 amino acid residues with a molecular weight of 33,714. LipP also has consensus motifs conserved in other cold-adapted lipases, i.e., Lipase 2 from Antarctic Moraxella TA144 (G. Feller, M. Thirty, J. L. Arpigny, and C. Gerday, DNA Cell Biol. 10:381-388, 1991) and the mammalian hormone-sensitive lipase (D. Langin, H. Laurell, L. S. Holst, P. Belfrage, and C. Holm, Proc. Natl. Acad. Sci. USA 90:4897-4901, 1993): a pentapeptide, GDSAG, containing the putative active-site serine and an HG dipeptide. LipP was purified from an extract of recombinant Escherichia coli C600 cells harboring a plasmid coding for the lipP gene. The enzyme showed a 1,3-positional specificity toward triolein. p-Nitrophenyl esters of fatty acids with short to medium chains (C4 and C6) served as good substrates. The enzyme was stable between pH 6 and 9, and the optimal pH for the enzymatic hydrolysis of tributyrin was around 8. The activation energies for the hydrolysis of p-nitrophenyl butyrate and p-nitrophenyl laurate were determined to be 11.2 and 7.7 kcal/mol, respectively, in the temperature range 5 to 35 degrees C. The enzyme was unstable at temperatures higher than 45 degrees C. The Km of the enzyme for p-nitrophenyl butyrate increased with increases in the assay temperature. The enzyme was strongly inhibited by Zn2+, Cu2+, Fe3+, and Hg2+ but was not affected by phenylmethylsulfonyl fluoride and bisnitrophenyl phosphate. Various water-miscible organic solvents, such as methanol and dimethyl sulfoxide, at concentrations of 0 to 30% (vol/vol) activated the enzyme.

Purification and characterization of a thermostable carboxylesterase from the thermoacidophilic eubacterium Bacillus acidocaldarius.

A thermostable carboxylesterase from the thermoacidophilic eubacterium Bacillus acidocaldarius was isolated, purified 1800-fold to homogeneity, and characterised. The apparent molecular mass was 36.5 +/- 2.5 kDa when determined by SDS/PAGE and 37.5 kDa when determined by analytical gel filtration, suggesting a monomeric structure. The pure enzyme regained activity on removal of SDS after SDS/PAGE. Several esterase activities were revealed in crude extracts by PAGE and activity staining, although only one was detected after SDS/PAGE and detergent removal. The esterase showed optimal activity at around 70 degrees C and pH 8, and was thermostable. p-Nitrophenyl esters of fatty acids from C2 to C12 were used as substrates; Vmax and Km values were determined at three different temperatures. The enzyme was able to hydrolyse tributyrylglycerol and trihexanoyl-glycerol dissolved in 0.8% acetonitrile, but neither lipase activity toward [14C]trioleoylglycerol nor proteolytic activity could be detected. Inactivation by diethyl p-nitrophenyl phosphate, by phenyl-methansulfonyl fluoride and physostigmine, and by diethylpyrocarbonate suggested that the enzyme contained a catalytic triad Ser-His-Asp/Glu in the active site, similar to that demonstrated for other serine-type enzymes. The amino acid composition and the sequence of 19 amino acid residues at the N-terminus were determined. These data, together with substrate preference and inhibition pattern, allowed us to classify this enzyme as a B-type carboxylesterase (EC 3.1.1.1).

Reaction of Lecithin Cholesterol Acyltransferase with Water-soluble Substrates

To separate the interfacial and catalytic reactions of lecithin cholesterol acyltransferase (LCAT), we carried out the first investigation of its reaction with water-soluble substrates. We used a continuous spectrophotometric assay for the hydrolysis of p-nitrophenyl esters of fatty acids to determine the chain length specificity of the enzyme and its modulation by anions and apolipoproteins in solution. By chemical modification of amino acid residues, we demonstrated that the active site serine and histidine residues participate in both the esterase and acyltransferase reactions but that cysteine residues are not involved in the esterase reaction. The kinetics of the LCAT reaction were measured for p-nitrophenyl esters of fatty acids having up to six (C-6) carbons in length. With increasing acyl chain lengths the optimal reaction rates occurred for the C-5 ester and Km and Vmax values decreased progressively, while the specificity constant, kcat/Km, increased. The same series of substrates and longer chain esters, up to C-16, were also reacted with LCAT in the presence of Triton X-100 in order to determine the general trends for the reaction rates as a function of chain length. The observed trends for the reaction rates and kinetic constants were attributed to an increasing binding affinity for the longer acyl chains in a large hydrophobic cavity, with a concomitant restriction in the motions of the substrates and a decreased probability for the correct positioning of the ester bond for hydrolysis, resulting in a decreased substrate turnover. Since the kinetics of the interfacial reactions of LCAT are very sensitive to the presence of anions and apolipoproteins, in particular apoA-I, we investigated the effects of these modulators on the reactions of LCAT in solution. Unlike the interfacial reactions, the hydrolysis of the p-nitrophenyl esters was not affected by 0.1 M concentrations of anions nor by water-soluble apolipoproteins (apoA-I, apoA-II, and apoCs). Thus the regulation of the activity of LCAT is mediated largely by the interfaces on which it acts.

Kinetic characterization of Escherichia coli outer membrane phospholipase A using mixed detergent-lipid micelles.

The substrate specificity of Escherichia coli outer membrane phospholipase A was analyzed in mixed micelles of lipid with deoxycholate or Triton X-100. Diglycerides, monoglycerides, and Tweens 40 and 85 in Triton X-100 are hydrolyzed at rates comparable to those of phospholipids and lysophospholipids. p-Nitrophenyl esters of fatty acids with different chain lengths and triglycerides are not hydrolyzed. The minimal substrate characteristics consist of a long acyl chain esterified to a more or less hydrophilic headgroup as is the case for the substrate monopalmitoylglycol. Binding occurs via the hydrocarbon chain of the substrate; diacyl compounds are bound three to five times better than monoacyl compounds. When acting on lecithins, phospholipase A1 activity is six times higher than phospholipase A2 activity or 1-acyl lysophospholipase activity. Activity on the 2-acyl lyso compound is about two times less than that on the 1-acyl lysophospholipid. The enzyme therefore has a clear preference for the primary ester bond of phospholipids. In contrast to phospholipase A1 activity, phospholipase A2 activity is stereospecific. Only the L isomer of a lecithin analogue in which the primary acyl chain was replaced by an alkyl ether group is hydrolyzed. The D isomer of this analogue is a competitive inhibitor, bound with the same affinity as the L isomer. On these ether analogues the enzyme shows the same preference for the primary acyl chain as with the natural diester phospholipids. Despite its broad specificity, the enzyme will initially act as a phospholipase A1 in the E. coli envelope where it is embedded in phospholipids.