Molecular characterisation of the gene encoding an esterase from Bacillus licheniformis sharing significant similarities with lipases

An esterase gene from the moderate thermophilic strain Bacillus licheniformis LCB40 was cloned and expressed in Escherichia coli. Comparison of the amino acid sequence of the esterase with those of known lipases and esterases showed the presence of the well-conserved Gly-X-Ser-X-Gly pentapeptide, with an alanine replacing the first glycine. This substitution has never been reported for an esterase but it is present in the lipases from Bacillus subtilis, Bacillus pumilus and Galactomyces candidum. The amino acid sequence showed similarities with lipases and with mammalian lecithin-cholesterol acyltranferases and no similarities with esterases. The enzyme activity of a crude extract from a recombinant Escherichia coli strain showed hydrolysis of p-nitrophenyl caprylate (pNPC8) as for esterases, but not of p-nitrophenyl palmitate (pNPC16) or olive oil such as for lipases. Thus, the enzyme displays the original property of associating the activity of an esterase with a primary sequence showing high similarity with lipases.     

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.     

Purification and properties of a novel extra-cellular thermotolerant metallolipase of Bacillus coagulans MTCC-6375 isolate

A novel extra-cellular lipase from Bacillus coagulans MTCC-6375 was purified 76.4-fold by DEAE anion exchange and Octyl Sepharose chromatography. The purified enzyme was found to be electrophoretically pure by denaturing gel electrophoresis and possessed a molecular mass of approximately 103 kDa. The lipase was optimally active at 45 degrees C and retained approximately 50% of its original activity after 20 min of incubation at 55 degrees C. The enzyme was optimally active at pH 8.5. Mg2+, Cu2+, Ca2+, Hg2+, Al3+, and Fe3+ at 1mM enhanced hydrolytic activity of the lipase. Interestingly, Hg2+ ions resulted in a maximal increase in lipase activity but Zn2+ and Co2+ ions showed an antagonistic effect on this enzyme. EDTA at 150 mM concentration inhibited the activity of lipase but Hg2+ or Al3+ (10mM) restored most of the activity of EDTA-quenched lipase. Phenyl methyl sulfonyl fluoride (PMSF, 15 mM) decreased 98% of original activity of lipase. The lipase was more specific to p-nitrophenyl esters of 8 (pNPC) and 16 (pNPP) carbon chain length esters. The lipase had a Vmax and Km of 0.44 mmol mg(-1)min(-1) and 28 mM for hydrolysis of pNPP, and 0.7 mmol mg(-1)min(-1) and 32 mM for hydrolysis of pNPC, respectively.     

New thermophilic and thermostable esterase with sequence similarity to the hormone-sensitive lipase family, cloned from a metagenomic library

A gene coding for a thermostable esterase was isolated by functional screening of Escherichia coli cells that had been transformed with fosmid environmental DNA libraries constructed with metagenomes from thermal environmental samples. The gene conferring esterase activity on E. coli grown on tributyrin agar was composed of 936 bp, corresponding to 311 amino acid residues with a molecular mass of 34 kDa. The enzyme showed significant amino acid similarity (64%) to the enzyme from a hyperthermophilic archaeon, Pyrobaculum calidifontis. An amino acid sequence comparison with other esterases and lipases revealed that the enzyme should be classified as a new member of the hormone-sensitive lipase family. The recombinant esterase that was overexpressed and purified from E. coli was active above 30 degrees C up to 95 degrees C and had a high thermal stability. It displayed a high degree of activity in a pH range of 5.5 to 7.5, with an optimal pH of approximately 6.0. The best substrate for the enzyme among the p-nitrophenyl esters (C(4) to C(16)) examined was p-nitrophenyl caproate (C(6)), and no lipolytic activity was observed with esters containing an acyl chain length of longer than 10 carbon atoms, indicating that the enzyme is an esterase and not a lipase.     

Overexpression of Serratia marcescens lipase in Escherichia coli for efficient bioresolution of racemic ketoprofen

Lipase from Serratia marcescens ECU1010 was cloned and overexpressed in E. coli. After optimization, the maximum lipase activities reached 5000–6000 U/l and this recombinant lipase could enantioselectively hydrolyze (S)-ketoprofen esters into (S)-ketoprofen. Among six alkyl esters of racemic ketoprofen investigated, this lipase showed the best enantioselectivity for the kinetic resolution of ketoprofen ethyl ester, with an ee_p (enantiomeric excess of product) of 91.6% and E-value of 63 obtained at 48.2% conversion. Twelve nonionic surfactants were tested for enhancing the enantioselectivity of this lipase in the bioresolution of ketoprofen ethyl ester. A very high E-value of 1084 was achieved, with an optical purity of >99% ee_p and a yield of 42.6% in the presence of 3% Brij 92V. Further studies showed that the selectivity of the lipase was improved with the increase of Brij 92V concentration. The substrate (ketoprofen ethyl ester) does not inhibit the lipase activity, while the product (S)-ketoprofen inhibits the lipase activity to some extent. These results indicate that the S. marcescens lipase is very useful for biocatalytic production of chiral profens such as (S)-ketoprofen.     

Lipase and Esterase Activities of Propionibacterium freudenreichii subsp. freudenreichii

The lipase and esterase activities of eight strains of dairy Propionibacterium freudenreichii subsp. freudenreichii were studied. A lipase activity was detected on whole cells and in the culture supernatant. The highest activity was expressed at 45°C and pH 6.8. An esterase activity was also detected in the culture medium. The electrophoresis of the intracellular fractions of the cells revealed from three to six different esterase activities. Two esterases were common to all the strains. The substrate specificity was dependent on each esterase, but no activity was revealed, in our experimental conditions, on ester substrates with a chain length longer than that of butyrate.     

Characterization and structural modeling of a new type of thermostable esterase from Thermotoga maritima

A bioinformatic screening of the genome of the hyperthermophilic bacterium Thermotoga maritima for ester-hydrolyzing enzymes revealed a protein with typical esterase motifs, though annotated as a hypothetical protein. To confirm its putative esterase function the gene (estD) was cloned, functionally expressed in Escherichia coli and purified to homogeneity. Recombinant EstD was found to exhibit significant esterase activity with a preference for short acyl chain esters (C4-C8). The monomeric enzyme has a molecular mass of 44.5 kDa and optimal activity around 95 degrees C and at pH 7. Its thermostability is relatively high with a half-life of 1 h at 100 degrees C, but less stable compared to some other hyperthermophilic esterases. A structural model was constructed with the carboxylesterase Est30 from Geobacillus stearothermophilus as a template. The model covered most of the C-terminal part of EstD. The structure showed an alpha/beta-hydrolase fold and indicated the presence of a typical catalytic triad consisting of a serine, aspartate and histidine, which was verified by site-directed mutagenesis and inhibition studies. Phylogenetic analysis showed that EstD is only distantly related to other esterases. A comparison of the active site pentapeptide motifs revealed that EstD should be grouped into a new family of esterases (Family 10). EstD is the first characterized member of this family.     

Esterase and lipase activity in Jatropha curcas L. seeds

Two new esterases (JEA and JEB) and a lipase (JL) were extracted from the seeds of Jatropha curas L. Lipase activity was only found during germination of the seeds and increased to a maximum after 4 days of germination. All enzymes were found to be most active in the alkaline range at around pH 8 and the purified (fractionated precipitation with ethanol and gel filtration) esterases were very stable at high temperatures. The molecular weight (SDS-PAGE) of both esterases was determined to be 21.6-23.5 kDa (JEA) and 30.2 kDa (JEB) and the isoelectric point was 5.7-6.1 for esterase JEA and 9.0 for esterase JEB. Most ions caused a negative influence on the activity of both esterases. Using p-nitrophenyl butyrate as a substrate JEA showed a K(m) of 0.02 mM and a v(max) of 0.26 micromol mg(-1) min(-1). Under the same conditions JEB showed a K(m) of 0.07 mM and a v(max) of 0.24 micromol mg(-1) min(-1). Both esterases hydrolyzed tributyrin, nitrophenyl esters up to a chain length of =C4 and naphtylesters up to a chain length =C6. In transesterification reactions, JL was found to be most active at very low water activities (0.2) and in high water activities, the lipase hydrolysed triglycerides into conversions above 80%. The lipase hydrolysed both short chain and long chain triglycerides at about the same rate but was inactive on alpha-methylbenzyl acetate. JL is a potentially useful biocatalyst in the hydrolysis of triglycerides in organic solvents.     

Extremely thermostable esterases from the thermoacidophilic euryarchaeon Picrophilus torridus

Two genes encoding esterases EstA and EstB of Picrophilus torridus were identified by the means of genome analysis and were subsequently cloned in Escherichia coli. PTO 0988, which is encoding EstA, consists of 579 bp, whereas PTO 1141, encoding EstB, is composed of 696 bp, corresponding to 192 aa and 231 aa, respectively. Sequence comparison revealed that both biocatalysts have low sequence identities (14 and 16%) compared to previously characterized enzymes. Detailed analysis suggests that EstA and EstB are the first esterases from thermoacidophiles not classified as members of the HSL family. Furthermore, the subunits with an apparent molecular mass of 22 and 27 kDa of the homotrimeric EstA and EstB, respectively, represent the smallest esterase subunits from thermophilic microorganisms reported to date. The recombinant esterases were purified by Ni²⁺ affinity chromatography, and the activity of the purified esterases was measured over a wide pH (pH 4.5–8.5) and temperature range (10–90°C). Highest activity of the esterases was measured at 70°C (EstA) and 55°C (EstB) with short pNP-esters as preferred substrates. In addition, esters of the non-steroidal anti-inflammatory drugs naproxen, ketoprofen, and ibuprofen are hydrolyzed by both EstA and EstB. Extreme thermostability was measured for both enzymes at temperatures as high as 90°C. The determined half-life (t _1/2) at 90°C was 21 and 10 h for EstA and EstB, respectively. Remarkable preservation of esterase activity in the presence of detergents, urea, and commonly used organic solvents complete the exceptional phenotype of EstA and EstB.     

A new esterase, belonging to hormone-sensitive lipase family, cloned from Rheinheimera sp. isolated from industrial effluent

The gene for esterase (rEst1) was isolated from a new species of genus Rheinheimera by functional screening of E. coli cells transformed with the pSMART/HaeIII genomic library. E. coli cells harboring the esterase gene insert could grow and produce clear halo zones on tributyrin agar. The rEst1 ORF consisted of 1,029 bp, corresponding to 342 amino acid residues with a molecular mass of 37 kDa. The signal P program 3.0 revealed the presence of a signal peptide of 25 amino acids. Esterase activity, however, was associated with a homotrimeric form of molecular mass 95 kDa and not with the monomeric form. The deduced amino acid sequence showed only 54% sequence identity with the closest lipase from Cellvibrio japonicus strain Ueda 107. Conserved domain search and multiple sequence alignment revealed the presence of an esterase/ lipase conserved domain consisting of a GXSXG motif, HGGG motif (oxyanion hole) and HGF motif, typical of the class IV hormone sensitive lipase family. On the basis of the sequence comparison with known esterases/ lipases, REst1 represents a new esterase belonging to class IV family. The purified enzyme worked optimally at 50 degrees C and pH 8, utilized pNP esters of short chain lengths, and showed best catalytic activity with p-nitrophenyl butyrate (C?), indicating that it was an esterase. The enzyme was completely inhibited by PMSF and DEPC and showed moderate organotolerance.     

Screening and its potential application of lipolytic activity from a marine environment: characterization of a novel esterase from <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> 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°C, respectively. rYli180 preferentially hydrolyzed p-nitrophenyl esters of fatty acids with short chain lengths of ≤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.     

Pancreatic cholesterol esterases. 3. Kinetic characterization of cholesterol ester resynthesis by the pancreatic cholesterol esterases

The ability of cholesterol esterase to catalyze the synthesis of cholesterol esters has been considered to be of limited physiological significance because of its bile salt requirements for activity, though detailed kinetic studies have not been reported. This study was performed to determine the taurocholate, pH, and substrate requirements for optimal cholesterol ester synthesis catalyzed by various pancreatic lipolytic enzymes, including the bovine 67- and 72-kDa cholesterol esterases, human 100-kDa cholesterol esterase, and human 52-kDa triglyceride lipase. In contrast to current beliefs, cholesterol esterase exhibits a bile salt independent as well as a bile salt dependent synthetic pathway. For the bovine pancreatic 67- and 72-kDa cholesterol esterases, the bile salt independent pathway is optimal at pH 6.0-6.5 and is stimulated by micromolar concentrations of taurocholate. For the bile salt dependent synthetic reaction for the 67-kDa enzyme, increasing the taurocholate concentration from 0 to 1.0 mM results in a progressive shift in the pH optimum from pH 6.0-6.5 to pH 4.5 or lower. In contrast, cholesterol ester hydrolysis by the 67-, 72-, and 100-kDa enzymes was characterized by pH optima from 5.5 to 6.5 at all taurocholate concentrations. Optimum hydrolytic activity for these three enzyme forms occurred with 10 mM taurocholate. Since hydrolysis is minimal at low taurocholate concentrations, the rate of synthesis actually exceeds hydrolysis when the taurocholate concentration is less than 1.0 mM. The 52-kDa enzyme exhibits very low cholesterol ester synthetic and hydrolytic activities, and for this enzyme both activities are bile salt independent. Thus, our data show that cholesterol esterase has both bile salt independent and bile salt dependent cholesterol ester synthetic activities and that it may catalyze the net synthesis of cholesterol esters under physiological conditions.     

Production and characterization of esterase and lipase from <Emphasis Type="Italic">Haloarcula marismortui</Emphasis>

The present study was conducted to investigate the capability of Haloarcula marismortui to synthesize esterases and lipases, and the effect of physicochemical conditions on the growth and the production of esterases and lipases. Finally, the effect of NaCl concentration and temperature on esterase and lipase activities was studied using intracellular crude extracts. In order to confirm the genomic prediction about the esterase and lipase synthesis, H. marismortui was cultured on a rich medium and the crude extracts (intra- or extracellular) obtained were assayed for both activities using p-nitrophenyl esters and triacylglycerides as substrates. Studies on the kinetics of growth and production of esterase and lipase of H. marismortui were performed, reaching a maximum growth rate of 0.053 h⁻¹ and maximal productions of intracellular esterase and lipase of 2.094 and 0.722 U l⁻¹ using p-nitrophenyl valerate and p-nitrophenyl laurate, respectively. Both enzymes were produced as growth-associated metabolites. The effects of temperature, pH, and NaCl concentration on the growth rate and production of enzymes were studied by using a Box–Behnken response surface design. The three response variables were significantly influenced by the physicochemical factors and an interaction effect between temperature and NaCl concentration was also evidenced. The surface response method estimated the following maximal values for growth rate and productions of esterase and lipase: 0.086 h⁻¹ (at 42.5°C, pH 7.4, and 3.6 mol l⁻¹ NaCl), 2.3 U l⁻¹ (at 50°C, pH 7.5, and 4.3 mol l⁻¹ NaCl), and 0.58 U l⁻¹ (at 50°C, pH 7.6, and 4.5 mol l⁻¹ NaCl), respectively. Esterases were active at different salt concentrations, showing two optimal activities (at 0.5 and 5 mol l⁻¹ NaCl), which suggested the presence of two different esterases. Interestingly, in the absence of salt, esterase retained 50% residual activity. Esterases and lipase activities were maximal at 45°C and inactive at 75°C. This study represents the first report evidencing the synthesis of esterase and lipase by H. marismortui.     

Lipase of Mucor pusillus

Lipase of Mucor pusillus NRRL 2543 was recovered with ammonium sulfate precipitation, gel filtration on Sephadex G-75, and anion-exchange chromatography on diethylaminoethyl-Sephadex A-50. Maximal glycerol ester hydrolase (lipase) activity was observed at pH 5.0 to 5.5 and 50 C when trioctanoin and olive oil were used as substrates. The enzyme also showed esterase activity; it hydrolyzed, with the exception of methyl butyrate, all methyl esters tested. A minimum chain length of six carbons appeared to be a requirement for esterase activity, which was maximal at about pH 5.5 with methyl dodecanoate (C(12)) as the substrate. Neither the glycerol ester hydrolase (lipase) nor the esterase activity of the enzyme appeared to be affected by thiol group inhibitors, chelating agents, and reducing compounds. On the other hand, hydrolysis of triolein and methyl dodecanoate was arrested to the same extent in the presence of diisopropyl fluorophosphate, which suggested the involvement of serine in the active center of the enzyme. The enzyme remained stable during a 30-day storage at - 10 C.     

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.     

Genome-wide cloning and characterization of microbial esterases

We have isolated putative esterase genes from various bacterial chromosomes. Thirty open reading frames predicted to encode esterases were randomly selected from 13 sequenced bacterial chromosomes and were cloned into an expression vector. The esterase activity of the resulting clones was tested on a tributyrin plate at different pH values and temperatures. Nine out of thirty tested clones exhibited significant tributyrin hydrolyzing activity. The enzyme S5 from the gene b0494 of Escherichia coli, the enzyme S12 from the gene STM0506 of Salmonella typhimurium, and the enzyme S28 from the gene AF1716 of Archaeoglobus fulgidus exhibited high activity at an alkaline pH range. The esterase S11 encoded by the gene PA3859 of Pseudomonas aeruginosa PAO1 and the esterase S21 from the gene SMc01033 of Sinorhizobium meliloti 1021, both showed a sharp increase in enzyme activity above pH 8.0. Furthermore, the enzymes S5, S12, S21, and S28 retained the esterase activity when they were incubated at 50 degrees C, suggesting that these enzymes are thermostable. Subsequent pH vs. activity and temperature vs. activity experiments with selected enzymes in a solution assay system confirmed the validity of the above data. The genome-wide exploration strategy of proteins provided valuable information on the esterases by revealing subtle biochemical differences between the esterases of different sources.     

Mycoplasma hyopneumoniae p65 surface lipoprotein is a lipolytic enzyme with a preference for shorter-chain fatty acids

Mycoplasma hyopneumoniae is the most significant bacterial pathogen of the respiratory tract of swine. p65 is an immunodominant surface lipoprotein of M. hyopneumoniae that is specifically recognized during disease. Analysis of the translated amino acid sequence of the gene encoding p65 revealed similarity to the GDSL family of lipolytic enzymes. To examine the lipolytic activity of p65, the gene was cloned and expressed in Escherichia coli after truncation of the prokaryotic lipoprotein signal sequence and mutagenesis of the mycoplasma TGA tryptophan codons. After treatment with thrombin, the recombinant glutathione S-transferase (GST)-p65 protein yielded a 66-kDa fusion protein cleavage product corresponding in size to the mature p65 protein. The esterase activity of recombinant GST-p65 was indicated by the formation of a cleared zone on tributyrin agar plates and the hydrolysis of p-nitrophenyl esters of caproate (pNPC) and p-nitrophenyl esters of palmitate (pNPP). Lipase activity was indicated by the hydrolysis of the artificial triglyceride 1,2-O-dilauryl-rac-glycero-3-glutaric acid resorufin ester. Using pNPC and pNPP as substrates, recombinant GST-p65 had optimal activity between pHs 9.2 and 10.2 and at a temperature higher than 39 degrees C. Calcium ions did not increase the activity of recombinant GST-p65. Rabbit anti-p65 antibodies inhibited the activity of recombinant GST-p65 and also inhibited the growth of M. hyopneumoniae in vitro. Examination of the kinetic parameters of recombinant GST-p65 for the hydrolysis of pNPC and pNPP indicated a preference for the shorter fatty acid chain of pNPC. The physiological and/or pathogenic role of mycoplasma lipolytic enzymes has not been determined, but they are likely to play an important role in mycoplasmas' nutritional requirements for long-chain fatty acids and may reduce the function of lung surfactants in mycoplasma-induced respiratory diseases. This is the first report of the lipolytic activity of a lipid-modified surface immunogen of a mycoplasma.     

Purification, characterization, and molecular cloning of organic-solvent-tolerant cholesterol esterase from cyclohexane-tolerant Burkholderia cepacia strain ST-200

Extracellular cholesterol esterase of Burkholderia cepacia strain ST-200 was purified from the culture supernatant. Its molecular mass was 37 kDa. The enzyme was stable at pH 5.5–12 and active at pH 5.5–6, showing optimal activity at pH 7.0 at 45°C. Relative to the commercially available cholesterol esterases, the purified enzyme was highly stable in the presence of various water-miscible organic solvents. The enzyme preferentially hydrolyzed long-chain fatty acid esters of cholesterol, except for that of cholesteryl palmitate. The enzyme exhibited lipolytic activity toward various p-nitrophenyl esters. The hydrolysis rate of p-nitrophenyl caprylate was enhanced 3.5- to 7.2-fold in the presence of 5–20% (vol/vol) water-miscible organic solvents relative to that in the absence of organic solvents. The structural gene encoding the cholesterol esterase was cloned and sequenced. The primary translation product was predicted to be 365 amino acid residues. The mature product is composed of 325 amino acid residues. The amino acid sequence of the product showed the highest similarity to the lipase LipA (87%) from B. cepacia DSM3959.     

Formation and splitting of esters in subterminal oxidation of dodecane by Fusarium lini

Summary Extracellular oxidation products having the same number of carbon atoms as the alkane that was oxidized were isolated from a Fusarium lini culture broth grown on n-dodecane. They were secondary isomeric alcohols, corresponding isomeric ketones and isomeric esters with 12 carbon atoms.Esterase activity in cell-free extracts of the fungus which was incubated on a p-nitrophenyl-acetate substrate increased with increasing temperatures and pH-values in the ranges 20–40°C and pH 6.0 to 8.0 respectively. The activity, when incubated on p-nitrophenyl-acetate,-laurate and-palmitate substrates, decreased with decreasing fatty acid chain lengths. When incubated with isomeric esters consisting of 12 carbon atoms, it was influenced by the ester linkage position in the chain. When the alcohol chain length in the ester increased from one to six carbon atoms, the esterase activity decreased. The same effect was observed when the chain length of the acid increased from two to six carbon atoms. Minimum esterase activity was reached when both the alcohol and the acid had a chain length of six carbon atoms.The view that all ketones produced during subterminal oxidation of alkanes by Fusarium lini and perhaps other members of Moniliales are further metabolized via ester intermediates is supported. A probable non-specific esterase or lipase catalyses the hydrolysis of the isomeric esters which are formed from the ketones.     

Purification and characterization of a novel extracellular lipase catalyzing hydrolysis of oleyl benzoate from Acinetobacter nov. sp. strain KM109

A new lipase (OBase) which efficiently hydrolyzes oleyl benzoate (OB) was found in the culture supernatant of Acinetobacter nov. sp. strain KM109, a new isolate growing in a minimum medium containing OB as the sole carbon source. OBase was purified to homogeneity with 213-fold purification and 0.8% yield. The molecular weight was estimated to be 62,000 +/- 1,000 by SDS-PAGE under denatured-reduced conditions and to be 50,000 +/- 1,000 by gel-filtration HPLC under native conditions; these findings indicate that OBase is a monomeric enzyme. The optimum temperature and pH of OBase were about 45 degrees C and pH 8. Temperature and pH stabilities were at or lower than 35 degrees C and in a range of pH 6-8, respectively. Purified OBase preferentially hydrolyzed p-nitrophenyl benzoate (pNPB) over p-nitrophenyl acetate (pNPA) or p-nitrophenyl caproate (pNPC) [pNPB/pNPA = 20 and pNPB/pNPC = 5.4], indicating that OBase has a high affinity for benzoyl esters. Partial amino-acid sequences of OBase fragments obtained after lysyl endopeptidase treatment showed no similarity with known proteins.