Might the kinetic behavior of hormone-sensitive lipase reflect the absence of the lid domain?

Hormone-sensitive lipase (HSL) is thought to contribute importantly to the mobilization of fatty acids from the triacylglycerols (TAGs) stored in adipocytes, providing the main source of energy in mammals. To investigate the HSL substrate specificity more closely, we systematically assessed the lipolytic activity of recombinant human HSL on solutions and emulsions of various vinyl esters and TAG substrates, using the pH-stat assay technique. Recombinant human HSL activity on solutions of partly soluble vinyl esters or TAG was found to range from 35 to 90% of the maximum activity measured with the same substrates in the emulsified state. The possible existence of a lipid-water interface due to the formation of small aggregates of vinyl esters or TAG in solution may account for the HSL activity observed below the solubility limit of the substrate. Recombinant human HSL also hydrolyzes insoluble medium- and long-chain acylglycerols such as trioctanoylglycerol, dioleoylglycerol, and olive oil, and can therefore be classified as a true lipase. Preincubation of the recombinant HSL with a serine esterase inhibitor such as diethyl p-nitrophenyl phosphate in 1:100 molar excess leads to complete HSL inhibition within 15 min. This result indicates that the catalytic serine of HSL is highly reactive and that it is readily accessible. Similar behavior was also observed with lipases with no lid domain covering their active site, or with a deletion in the lid domain. The 3-D structure of HSL, which still remains to be determined, may therefore lack the lid domain known to exist in various other lipases.     

Use of an inhibitor to identify members of the hormone-sensitive lipase family

Hormone-sensitive lipase (HSL) contributes importantly to the mobilization of fatty acids from the triacylglycerols stored in adipocytes, which provide the main source of energy in mammals. On the basis of amino acid sequence alignments and three-dimensional structures, this enzyme was previously found to be a suitable template for defining a family of serine carboxylester hydrolases. In this study, the HSL family members are characterized rather on the basis of their inhibition by 5-methoxy-3-(4-phenoxyphenyl)-3H-[1,3,4]oxadiazol-2-one (compound 7600). This compound inhibits mammalian HSL as well as other HSL family members, such as EST2 from the thermophilic eubacterium Alicyclobacillus acidocaldarius and AFEST from the hyperthermophilic archaeon Archaeoglobus fulgidus. Various carboxylester hydrolases that are not members of the HSL family were found not to be inhibited by compound 7600 under the same experimental conditions. These include nonlipolytic hydrolases such as Torpedo californica acetylcholinesterase and pig liver esterase, as well as lipolytic hydrolases such as human pancreatic lipase, dog gastric lipase, Thermomyces lanuginosus lipase, and Bacillus subtilis LipA. When vinyl esters were used as substrates, the residual activity of HSL, AFEST, and EST2 decreased with an increase in compound 7600 concentration in the incubation mixture. The inhibitor concentration at which the enzyme activity decreased to 50% after incubation for 5 min was 70, 20, and 15 nM with HSL, AFEST, and EST2, respectively. Treating EST2 and AFEST with the inhibitor resulted in an increase in the molecular mass, as established by performing matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis. This increase in the molecular mass, which corresponds approximately to the molecular mass of the inhibitor, indicates that a covalent enzyme-inhibitor complex has been formed. Surface-enhanced laser desorption ionization time-of-flight mass spectrometry analysis of a trypsin digest of AFEST treated with the inhibitor or not treated showed the occurrence of an increase in the molecular masses of the "GESAGG"-containing peptide, which is compatible with the formation of a covalent complex with the inhibitor.     

超嗜热羧酸酯酶的性质和应用研究

摘要 来源于超嗜热菌的超嗜热羧酸酯酶,结构上与激素敏感性脂肪酶(HSL)家族近似,属于α/β-水解酶,但是其空间结构比HSL更加紧密而有韧性,具有很强的热稳定性。性质研究表明,温度和有机溶剂对超嗜热羧酸酯酶的活性和对映选择性影响显著;其最适底物一般是对硝基苯酯,部分酯酶的基因含有GGGX基序,能够水解叔醇酯结构。由于超嗜热羧酸酯酶的独特结构和性质,其应用潜力巨大,尤其在拆分手性的外消旋酯方面独具优势。     

Lipase-catalysed hydrolysis of short-chain substrates in solution and in emulsion: a kinetic study.

We have studied the enzymatic hydrolysis of solutions and emulsions of vinyl propionate, vinyl butyrate and tripropionin by lipases of various origin and specificity. Kinetic studies of the hydrolysis of short-chain substrates by microbial triacylglycerol lipases from Rhizopus oryzae, Mucor miehei, Candida rugosa, Candida antarctica A and by (phospho)lipase from guinea-pig pancreas show that these lipolytic enzymes follow the Michaelis-Menten model. Surprisingly, the activity against solutions of tripropionin and vinyl esters ranges from 70% to 90% of that determined against emulsions. In contrast, a non-hyperbolic (sigmoidal) dependence of enzyme activity on ester concentration is found with human pancreatic lipase, triacylglycerol lipase from Humicola lanuginosa (Thermomyces lanuginosa) and partial acylglycerol lipase from Penicillium camembertii and the same substrates. In all cases, no abrupt jump in activity (interfacial activation) is observed at substrate concentration corresponding to the solubility limit of the esters. Maximal lipolytic activity is always obtained in the presence of emulsified ester. Despite progress in the understanding of structure-function of lipases, interpretation of the mode of action of lipases active against solutions of short-chain substrates remains difficult. Actually, it is not known whether these enzymes, which possess a lid structure, are in open or/and closed conformation in the bulk phase and whether the opening of the lid that gives access to the catalytic triad is triggered by interaction of the enzyme molecule with monomeric substrates or/and multimolecular aggregates (micelles) both present in the bulk phase. From the comparison of the behaviour of lipases used in this study which, in some cases, follow the Michaelis-Menten model and, in others, deviate from classical kinetics, it appears that the activity of classical lipases against soluble short-chain vinyl esters and tripropionin depends not only on specific interaction with single substrate molecules at the catalytic site of the enzyme but also on physico-chemical parameters related to the state of association of the substrate dispersed in the aqueous phase. It is assumed that the interaction of lipase with soluble multimolecular aggregates of tripropionin or short-chain vinyl esters or the formation of enzyme-substrate mixed micelles with ester bound to lipase, might represent a crucial step that triggers the structural transition to the open enzyme conformation by displacement of the lid.     

Ser649 and Ser650 Are the Major Determinants of Protein Kinase A-Mediated Activation of Human Hormone-Sensitive Lipase against Lipid Substrates

Background

Hormone-sensitive lipase (HSL) is a key enzyme in the mobilization of fatty acids from stored triacylglycerols. Its activity is regulated by reversible protein phosphorylation. In rat HSL Ser563, Ser659 and Ser660 have been shown to be phosphorylated by protein kinase A (PKA) in vitro as well as in vivo.

Methodology/Principal Findings

In this study we employed site-directed mutagenesis, in vitro phosphorylation and mass spectrometry to show that in vitro phosphorylation of human HSL by PKA occurs primarily on Ser649 and Ser650 (Ser659 and Ser660 in rat HSL). The wild type enzyme and four mutants were expressed in C-terminally His-tagged form in Sf9 insect cells and purified to homogeneity. HSL variants in which Ser552 and/or Ser554 were mutated to Ala or Glu retained both lipolytic and non-lipolytic activity and were phosphorylated by PKA and activated to a similar extent as the wild type enzyme. ³²P-labeling studies revealed that the bulk of the phosphorylation was on the Ser649/Ser650 site, with only a minor phosphorylation of Ser552 and Ser554. MS/MS analysis demonstrated that the peptide containing Ser649 and Ser650 was primarily phosphorylated on Ser650. The mutant lacking all four serines had severely reduced lipolytic activity, but a lesser reduction in non-lipolytic activity, had S_0.5 values for p-nitrophenol butyrate and triolein comparable to those of wild type HSL and was not phosphorylated by PKA. PKA phosphorylation of the wild type enzyme resulted in an increase in both the maximum turnover and S_0,5 using the TO substrate.

Conclusions

Our results demonstrate that PKA activates human HSL against lipid substrates in vitro primarily through phosphorylation of Ser649 and Ser650. In addition the results suggest that Ser649 and Ser650 are located in the vicinity of a lipid binding region and that PKA phosphorylation controls the accessibility of this region.     

Interfacial reaction dynamics and acyl-enzyme mechanism for lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters

The fatty acyl (lipid) p-nitrophenyl esters p-nitrophenyl caprylate, p-nitrophenyl laurate and p-nitrophenyl palmitate that are incorporated at a few mol % into mixed micelles with Triton X-100 are substrates for bovine milk lipoprotein lipase. When the concentration of components of the mixed micelles is approximately equal to or greater than the critical micelle concentration, time courses for lipoprotein lipase-catalyzed hydrolysis of the esters are described by the integrated form of the Michaelis-Menten equation. Least square fitting to the integrated equation therefore allows calculation of the interfacial kinetic parameters Km and Vmax from single runs. The computational methodology used to determine the interfacial kinetic parameters is described in this paper and is used to determine the intrinsic substrate fatty acyl specificity of lipoprotein lipase catalysis, which is reflected in the magnitude of kcat/Km and kcat. The results for interfacial lipoprotein lipase catalysis, along with previously determined kinetic parameters for the water-soluble esters p-nitrophenyl acetate and p-nitrophenyl butyrate, indicate that lipoprotein lipase has highest specificity for the substrates that have fatty acyl chains of intermediate length (i.e. p-nitrophenyl butyrate and p-nitrophenyl caprylate). The fatty acid products do not cause product inhibition during lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters that are contained in Triton X-100 micelles. The effects of the nucleophiles hydroxylamine, hydrazine, and ethylenediamine on Km and Vmax for lipoprotein lipase catalyzed hydrolysis of p-nitrophenyl laurate are consistent with trapping of a lauryl-lipoprotein lipase intermediate. This mechanism is confirmed by analysis of the product lauryl hydroxamate when hydroxylamine is the nucleophile. Hence, lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters that are contained in Triton X-100 micelles occurs via an interfacial acyl-lipoprotein lipase mechanism that is rate-limited by hydrolysis of the acyl-enzyme intermediate.     

Lipolytic activity from Halobacteria: screening and hydrolase production

Strains of Halobacteria from an Algerian culture collection were screened for their lipolytic activity against p-nitrophenyl butyrate (PNPB) and p-nitrophenyl palmitate (PNPP). Most strains were active on both esters and 12% hydrolyzed olive oil. A strain identified as Natronococcus sp. was further studied. It grew optimally at 3.5 M NaCl, pH 8 and 40 degrees C. An increase in temperature shifted the optimum salt concentration range for growth from a wider range of 2-4 M, obtained at 25-30 degrees C, to a narrower range of 3.5-4 M, obtained at 35-40 degrees C. At 45 degrees C the optimum salt concentration was 2 M. These results show a clear correlation between salt and temperature requirement. The optimum conditions for the production of hydrolytic activity during growth were: 3.5 M NaCl and pH 8 for PNPB hydrolytic activity and 4 M NaCl and pH 7.5 for PNPP hydrolytic activity; both at 40 degrees C. The clear supernatant of cells grown at 4 M NaCl showed olive oil hydrolysis activity (in presence of 4 M NaCl) demonstrating the occurrence of a lipase activity in this strain. To our knowledge, this is the first report of a lipase activity at such high salt concentration.     

Continuous monitoring of cholesterol oleate hydrolysis by hormone-sensitive lipase and other cholesterol esterases

Hormone-sensitive lipase (HSL) contributes importantly to the hydrolysis of cholesteryl ester in steroidogenic tissues, releasing the cholesterol required for adrenal steroidogenesis. HSL has broad substrate specificity, because it hydrolyzes triacylglycerols (TAGs), diacylglycerols, monoacylglycerols, and cholesteryl esters. In this study, we developed a specific cholesterol esterase assay using cholesterol oleate (CO) dispersed in phosphatidylcholine and gum arabic by sonication. To continuously monitor the hydrolysis of CO by HSL, we used the pH-stat technique. For the sake of comparison, the hydrolysis of CO dispersion was also tested using other cholesteryl ester-hydrolyzing enzymes. The specific activities measured on CO were found to be 18, 100, 27, and 3 micromol/min/mg for HSL, cholesterol esterase from Pseudomonas species, Candida rugosa lipase-3, and cholesterol esterase from bovine pancreas, respectively. The activity of HSL on CO is approximately 4- to 5-fold higher than on long-chain TAGs. In contrast, with all other enzymes tested, the rates of TAG hydrolysis were higher than those of CO hydrolysis. The relatively higher turnover of HSL on CO observed in vitro adds further molecular insight on the physiological importance of HSL in cholesteryl ester catabolism in vivo. Thus, HSL could be considered more as a cholesteryl ester hydrolase than as a TAG lipase.     

Lipase-catalysed hydrolysis of short-chain substrates in solution and in emulsion: a kinetic study

We have studied the enzymatic hydrolysis of solutions and emulsions of vinyl propionate, vinyl butyrate and tripropionin by lipases of various origin and specificity. Kinetic studies of the hydrolysis of short-chain substrates by microbial triacylglycerol lipases from Rhizopus oryzae, Mucor miehei, Candida rugosa, Candida antarctica A and by (phospho)lipase from guinea-pig pancreas show that these lipolytic enzymes follow the Michaelis–Menten model. Surprisingly, the activity against solutions of tripropionin and vinyl esters ranges from 70% to 90% of that determined against emulsions. In contrast, a non-hyperbolic (sigmoidal) dependence of enzyme activity on ester concentration is found with human pancreatic lipase, triacylglycerol lipase from Humicola lanuginosa (Thermomyces lanuginosa) and partial acylglycerol lipase from Penicillium camembertii and the same substrates. In all cases, no abrupt jump in activity (interfacial activation) is observed at substrate concentration corresponding to the solubility limit of the esters. Maximal lipolytic activity is always obtained in the presence of emulsified ester. Despite progress in the understanding of structure–function of lipases, interpretation of the mode of action of lipases active against solutions of short-chain substrates remains difficult. Actually, it is not known whether these enzymes, which possess a lid structure, are in open or/and closed conformation in the bulk phase and whether the opening of the lid that gives access to the catalytic triad is triggered by interaction of the enzyme molecule with monomeric substrates or/and multimolecular aggregates (micelles) both present in the bulk phase. From the comparison of the behaviour of lipases used in this study which, in some cases, follow the Michaelis–Menten model and, in others, deviate from classical kinetics, it appears that the activity of classical lipases against soluble short-chain vinyl esters and tripropionin depends not only on specific interaction with single substrate molecules at the catalytic site of the enzyme but also on physico-chemical parameters related to the state of association of the substrate dispersed in the aqueous phase. It is assumed that the interaction of lipase with soluble multimolecular aggregates of tripropionin or short-chain vinyl esters or the formation of enzyme–substrate mixed micelles with ester bound to lipase, might represent a crucial step that triggers the structural transition to the open enzyme conformation by displacement of the lid.     

Purification of rat kidney carboxylesterase and its comparison with other tissue esterases

Carboxylesterase was purified from rat kidney in an electrophoretically homogeneous form by acetone precipitation, followed by successive chromatographies on DEAE-cellulose and hydroxyapatite and then isoelectric focusing. The purified enzyme catalyzed the hydrolyses of monoacylglycerols and short-chain triacylglycerols, such as tributyrin, but not the hydrolysis of long-chain triacylglycerol. Its optimum pH with methyl butyrate as a substrate was 8.0. The relation of its activity to the methyl butyrate concentration differed from those for pancreatic lipase and liver esterase, and also from those for lipolytic enzymes from various other tissues. The relations of methyl butyrate-hydrolyzing activity with methyl butyrate concentration were compared among various carboxylester hydrolyzing enzymes. Based on the results, these enzymes were classified into four classes.     

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.     

Non-lipolytic and lipolytic sequence-related carboxylesterases: A comparative study of the structure–function relationships of rabbit liver esterase 1 and bovine pancreatic bile-salt-activated lipase

To differentiate esterases from lipases at the structure–function level, we have compared the kinetic properties and structural features of sequence-related esterase 1 from rabbit liver (rLE) and bile-salt-activated lipase from bovine pancreas (bBAL). In contrast to rLE, bBAL hydrolyses water-insoluble medium and long chain esters as vinyl laurate, trioctanoin and olive oil. Conversely, rLE and bBAL are both active on water-soluble short chain esters as vinyl acetate, vinyl propionate, vinyl butyrate, tripropionin, tributyrin and p-nitrophenyl butyrate. However, the enzymes show distinctive kinetic behaviours. rLE displays maximal activity at low substrate concentration, below the critical micelle concentration, whereas bBAL acts preferencially on emulsified esters, at concentration exceeding the solubility limit. Comparison of the 3D structures of rLE and bBAL shows, in particular, that the peptide loop at positions 116–123 in bBAL is deleted in rLE. This peptide segment interacts with a bile salt molecule thus inducing a conformational transition which gives access to the active site. Inhibition studies and manual docking of a bulky ester molecule as vinyl laurate in the catalytic pocket of rLE and bBAL show that the inability of the esterase to hydrolyse large water-insoluble esters is not due to steric hindrance. It is hypothesized that esterases lack specific hydrophobic structures involved both in the stabilization of the lipase–lipid adsorption complex at interfaces and in the spontaneous transfer of a single substrate molecule from interface to the catalytic site.     

Crystal structure and enhanced activity of a cutinase‐like enzyme from Cryptococcus sp. strain S‐2

The structural and enzymatic characteristics of a cutinase‐like enzyme (CLE) from Cryptococcus sp. strain S‐2, which exhibits remote homology to a lipolytic enzyme and a cutinase from the fungus Fusarium solani (FS cutinase), were compared to investigate the unique substrate specificity of CLE. The crystal structure of CLE was solved to a 1.05 Å resolution. Moreover, hydrolysis assays demonstrated the broad specificity of CLE for short and long‐chain substrates, as well as the preferred specificity of FS cutinase for short‐chain substrates. In addition, site‐directed mutagenesis was performed to increase the hydrolysis activity on long‐chain substrates, indicating that the hydrophobic aromatic residues are important for the specificity to the long‐chain substrate. These results indicate that hydrophobic residues, especially the aromatic ones exposed to solvent, are important for retaining lipase activity. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Synthesis and characterization of a new cutinase substrate, 4-nitrophenyl (16-methyl sulfone ester) hexadecanoate

Phytopathogenic fungi penetrate plants by breaking down the cuticular barrier with cutinase. Cutinases are extracellular hydrolytic enzymes that degrade cutin, a polyester composed of hydroxy and epoxy fatty acids. Until now, cutinase has been recognized by its ability to release labeled cutin monomers or by a non-specific esterase assay based on the hydrolysis of p-nitrophenyl esters of short fatty acids. In this work, an insoluble p-nitrophenyl derivative was synthesized and purified, and its structure was determined to be 4-nitrophenyl (16-methyl sulfone ester) hexadecanoate (pNMSEH) by nuclear magnetic resonance (H+ NMR) analysis. pNMSEH was tested as a new cutinase substrate with Pseudomonas mandocino cutinase and porcine liver esterase. While a linear release over time of p-nitrophenol (pNP) was recorded in the presence of cutinase, no response was obtained with the esterase. The calculated kinetic parameters of pNMSEH hydrolysis by cutinase revealed a high specificity (Km=1.8mM), albeit a low catalytic rate (Vmax=10.5 micromol min(-l)l(-1)). This new synthetic substrate may be helpful for detecting and assaying cutinase activity in mixed solutions, such as crude fungal extracellular extracts.     

A thermoactive uropygial esterase from chicken: purification, characterisation and synthesis of flavour esters

A lipolytic activity was located in the chicken uropygial glands, from which a carboxylesterase (CUE) was purified. Pure CUE has an apparent molecular mass of 50 kDa. The purified esterase displayed its maximal activity (200 U/mg) on short-chain triacylglycerols (tributyrin) at a temperature of 50°C. No significant lipolytic activity was found when medium chain (trioctanoin) or long chain (olive oil) triacylglycerols were used as substrates. The enzyme retained 75% of its maximal activity when incubated during 2h at 50°C. The NH(2)-terminal amino acid sequence showed similarities with the esterase purified recently from turkey pharyngeal tissue. Esterase activity remains stable after its incubation during 30 min in presence of organic solvents such as hexane or butanol. CUE is a serine enzyme since it was inactivated by phenylmethanesulphonyl fluoride (PMSF), a serine-specific inhibitor. The purified enzyme, which tolerates the presence of some organic solvent and a high temperature, can be used in non-aqueous synthesis reactions. Hence, the uropygial esterase immobilised onto CaCO(3) was tested to produce the isoamyl and the butyl acetate (flavour esters). Reactions were performed at 50°C in presence of hexane. High synthesis yields of 91 and 67.8% were obtained for isoamyl and butyl acetate, respectively.     

Expression and characterization of the protein Rv1399c from Mycobacterium tuberculosis. A novel carboxyl esterase structurally related to the HSL family.

The Mycobacterium tuberculosis genome contains an unusually high number of proteins involved in the metabolism of lipids belonging to the Lip family, including various nonlipolytic and lipolytic hydrolases. Driven by a structural genomic approach, we have biochemically characterized the Rv1399c gene product, LipH, previously annotated as a putative lipase. Rv1399c was overexpressed in E. coli as inclusion bodies and refolded. Rv1399c efficiently hydrolyzes soluble triacylglycerols and vinyl esters. It is inactive against emulsified substrate and its catalytic activity is strongly inhibited by the diethyl paranitrophenyl phosphate (E600). These kinetic behaviors unambiguously classify Rv1399c as a nonlipolytic rather than a lipolytic hydrolase. Sequence alignment reveals that this enzyme belongs to the alpha/beta hydrolase fold family and shares 30-40% amino acid sequence identity with members of the hormone-sensitive lipase subfamily. A model of Rv1399c derived from homologous three-dimensional structures reveals a canonical catalytic triad (Ser162, His290 and Asp260) located at the bottom of a solvent accessible pocket lined by neutral or charged residues. Based on this model, kinetic data of the Arg213Ala mutant partially explain the role of the guanidinium moiety, located close to His290, to confer an unusual low pH shift of the catalytic histidine in the wild type enzyme. Overall, these data identify Rv1399c as a new nonlipolytic hydrolase from M. tuberculosis and we thus propose to reannotate its gene product as NLH-H.     

Comparing the effect of immobilization methods on the activity of lipase biocatalysts in ester hydrolysis

The activity of various lipases was compared, in both free and immobilized forms, using the kinetics of the hydrolysis reaction of p-nitrophenyl butyrate, which was followed with in situ UV/Vis diode array spectrophotometry. Several enzymes were used to catalyze the reaction, namely Candida antarctica lipase B and Fusarium solani pisi cutinase wildtype and three single-mutation variants. The enzymes were tested in three different forms: free, immobilized as cross-linked aggregates and supported on zeolite NaY. A simple kinetic model was used to allow a quantitative comparison of the behavior of the different catalysts. It was concluded that although immobilization reduces the activity of the enzyme, the zeolite offers a much higher specific activity when compared to the cross-linked aggregates, thus supplying a heterogeneous catalyst with promising catalytic properties.     

Cloning and characterization of the lipase and lipase activator protein from Vibrio vulnificus CKM-1

The gene (lipA) encoding the extracellular lipase and its downstream gene (lipB) from Vibrio vulnificus CKM-1 were cloned and sequenced. Nucleotide sequence analysis and alignments of amino acid sequences suggest that Lip Ais a member of bacterial lipase family I.1 and that LipB is a lipase activator of LipA. The active LipA was produced in recombinant Escherichia coli cells only in the presence of the lipB. In the hydrolysis of p-nitrophenyl esters and triacylglycerols, using the reactivated LipA, the optimum chain lengths for the acyl moiety on the substrate were C14 for ester hydrolysis and C10 to C12 for triacylglycerol hydrolysis.     

Novel metagenome-derived carboxylesterase that hydrolyzes β-lactam antibiotics

It has been proposed that family VIII carboxylesterases and class C β-lactamases are phylogenetically related; however, none of carboxylesterases has been reported to hydrolyze β-lactam antibiotics except nitrocefin, a nonclinical chromogenic substrate. Here, we describe the first example of a novel carboxylesterase derived from a metagenome that is able to cleave the amide bond of various β-lactam substrates and the ester bond of p-nitrophenyl esters. A clone with lipolytic activity was selected by functional screening of a metagenomic library using tributyrin agar plates. The sequence analysis of the clone revealed the presence of an open reading frame (estU1) encoding a polypeptide of 426 amino acids, retaining an S-X-X-K motif that is conserved in class C β-lactamases and family VIII carboxylesterases. The gene was overexpressed in Escherichia coli, and the purified recombinant protein (EstU1) was further characterized. EstU1 showed esterase activity toward various chromogenic p-nitrophenyl esters. In addition, it exhibited hydrolytic activity toward nitrocefin, leading us to investigate whether EstU1 could hydrolyze β-lactam antibiotics. EstU1 was able to hydrolyze first-generation β-lactam antibiotics, such as cephalosporins, cephaloridine, cephalothin, and cefazolin. In a kinetic study, EstU1 showed a similar range of substrate affinities for both p-nitrophenyl butyrate and first-generation cephalosporins while the turnover efficiency for the latter was much lower. Furthermore, site-directed mutagenesis studies revealed that the catalytic triad of EstU1 plays a crucial role in hydrolyzing both ester bonds of p-nitrophenyl esters and amide bonds of the β-lactam ring of antibiotics, implicating the predicted catalytic triad of EstU1 in both activities.