Purification and characterization of the tween-hydrolyzing esterase of Mycobacterium smegmatis.

An esterase hydrolyzing Tween 80 (polyoxyethylene sorbitan monooleate) was purified from sonicated cell lysates of Mycobacterium smegmatis ATCC 14468 by DEAE-cellulose, Sephadex G-150, phenyl Sepharose, and diethyl-(2-hydroxypropyl) aminoethyl column chromatography and by subsequent preparative polyacrylamide gel electrophoresis. The molecular weight was estimated to be 36,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 41,000 by gel filtration on a Sephadex G-150 column. The esterase contained a single polypeptide. The esterase was stable to heat treatment at 100 degrees C and to a wide range of pH. The temperature and pH optima for the hydrolysis of Tween 80 were 50 degrees C and 8.3, respectively. The esterase had a narrow substrate specificity; it exhibited a high activity only on compounds having both polyoxyethylene and fatty acyl moieties, such as Tweens. Monoacylglyceride was hydrolyzed more slowly by this esterase and this enzyme exhibited a nonspecific esterase activity on p-nitrophenyl acyl esters, especially those having short chain acyl moieties. The Km and Vmax were 19.2 mM and 1,670 mumol/min per mg of protein for Tween 20, 6.6 mM and 278 mumol/min per mg of protein for Tween 80, and 0.25 mM and 196 mumol/min per mg of protein for p-nitrophenyl acetate, respectively. Observations of the effects of various chemical modifications on the activity of the esterase indicated that tyrosine, histidine, arginine, and methionine (with tryptophan) residues may be active amino acids which play important roles in the expression of Tween 80-hydrolyzing activity of the enzyme.     

Mycobacteriocins produced by rapidly growing mycobacteria are Tween-hydrolyzing esterases.

Smegmatocin, a protein produced by Mycobacterium smegmatis ATCC 14468, was found to have an esterase activity, hydrolyzing Tween 80, polyoxyethylene sorbitan monooleate, added to the assay medium for various "bacteriocins" from mycobacteria. Because M. diernhoferi ATCC 19340 (indicator strain for smegmatocin) is highly susceptible to oleic acid and smegmatocin requires Tween 80 for manifestation of its anti-M. diernhoferi activity, it is likely that smegmatocin-mediated antimicrobial action is caused by oleic acid generated by hydrolysis of Tween 80 by the inherent esterase action of smegmatocin. Other mycobacteriocins from rapidly growing mycobacteria also have inherent esterase activity against Tween 80 and require Tween 80 for expression of antimycobacterial action. Smegmatocin was found to hydrolyze various polyoxyethylene (sorbitan) fatty acyl esters but not sorbitan monooleate and glyceryl esters.     

Studies on sterol-ester hydrolase from Fusarium oxysporum. I. Partial purification and properties.

1. A search for a long chain fatty acyl sterol-ester hydrolase in microorganisms led to the isolation from soil of five strains belonging to Fusarium sp. which produced strong activity in the culture medium. 2. The cholesterol esterase from Fusarium oxysporum IGH-2 was purified about 270-fold by means of CaCl2 precipitation and Sephadex G-75 column chromatography. 3. The cholesterol esterase was activated by adekatol and Triton X-100. It was inhibited by lecithin and lysolecithin, and completely inactivated by heat treatment (60 degrees C for 30 min, at pH 7.0). 4. The optimum pH of the enzyme was found to be around 7.0. 5. Among various cholesterol esters tested, cholesterol linoleate was the most suitable substrate. 6. Cholesterol esters in serum were also hydrolyzed by this enzyme.     

Significance of lipoidal estradiol in human mammary tumors

Incubations of p-nitrophenyl fatty acyl esters and estradiol-17 beta fatty acyl 17-esters with porcine esterase, human mammary tumor cytosol and rat uterine cytosol leads to ester hydrolysis of compounds with short chain fatty acids. Esters with long chain fatty acids show no hydrolysis except in the presence of Tween 80. Short chain fatty acid esters have a higher binding potency to the estrogen receptor than long chain fatty acid esters. Extraction of the nuclear receptor peak sedimenting at 4.6S and identification of the steroid showed that about 90% of the radioactivity was associated with estradiol and only 10% with estradiol esters. These studies show that estradiol fatty acyl esters act as a storage form from which estradiol is released by enzymatic hydrolysis.     

A retinyl ester hydrolase activity intrinsic to the brush border membrane of rat small intestine.

Retinol esterified with long-chain fatty acids is a common dietary source of vitamin A. Hydrolysis of these esters in the lumen of the small intestine is required prior to absorption. Bile salt-stimulated retinyl esterase activity was present with purified rat intestinal brush border membrane, with the maximum rate of ester hydrolysis at approximately pH 8, the physiological luminal pH. Taurocholate, a trihydroxy bile salt, stimulated hydrolysis of short-chain fatty acyl retinyl esters more than hydrolysis of long-chain fatty acyl esters. Deoxycholate, a dihydroxy bile salt, primarily stimulated hydrolysis of long-chain esters. Calculated Kms of 0.74 microM for retinyl palmitate (16:0) hydrolysis and 9.6 microM for retinyl caproate (6:0) hydrolysis suggested the presence of two separate activities. Consistent with that, the activity responsible for retinyl caproate hydrolysis could be inactivated to a greater degree than retinyl palmitate hydrolysis by preincubation of the brush border membrane at 37 degrees C for extended times. Brush border membrane from animals who had undergone common duct ligation 48 h prior to tissue collection showed little ability to hydrolyze retinyl caproate but retained 70% of retinyl palmitate hydrolytic activity, compared to sham-operated controls. Thus, two distinguishable retinyl esterase activities were recovered with purified brush border membranes. One apparently originated from the pancreas, was stimulated by trihydroxy bile salts, and preferentially hydrolyzed short-chain retinyl esters, properties similar to cholesterol ester hydrolase, known to bind to the brush border. The other was intrinsic to the brush border, stimulated by both trihydroxy and dihydroxy bile salts, and preferentially hydrolyzed long-chain retinyl esters, providing the majority of activity of the brush border against dietary retinyl esters.     

Characterization of an acyl-coenzyme a thioesterase associated with the envelope of spinach chloroplasts

The enzymic hydrolysis of acyl-coenzyme A occurs in intact and purified chloroplasts. The different components of spinach chloroplasts were separated after a slight osmotic shock and the purified envelope membranes were shown to be the site of very active acyl-CoA thioesterase activity (EC 3.1.2.2.). The enzyme, which had a pH optimum of 9.0, was not affected by sulfhydryl reagents or by serine esterase inhibitors. However, the acyl-CoA thioesterase was strongly inhibited by unsaturated fatty acids, especially oleic acid, at concentrations above 100 micromolar. In marked contrast, saturated fatty acids had only a slight effect on the thioesterase activity. Substrate specificities showed that the velocity of the reaction increased with the chain length of the substrate from decanoyl-CoA to myristoyl-CoA and then decreased with the chain length from myristoyl-CoA to stearoyl-CoA. Interestingly, oleoyl-CoA was only slowly hydrolyzed. These results suggest that the envelope acyl-CoA thioesterase coupled with an envelope acyl-CoA synthetase may be involved in a switching system which indirectly allows acyl transfer from acyl carrier protein derivatives to unsaturated acyl-CoA derivatives and ensures the predominance of unsaturated 18 carbon fatty acids in plants. Furthermore, the position of both acyl-CoA thioesterase and synthetase in the envelope membranes suggest that these two enzymes may be involved in the transport of oleic acid from the stroma phase to the cytosol compartment of the leaf cell.     

Production and activity of extracellular lipase from Luteibacter sp.

Microbial lipases are widely used in industrial applications due to their versatility, and the characterization of new lipase-producing microorganisms could provide new sources of these enzymes, with different specificities and better activities. In this context, we have improved lipase production by Luteibacter sp. by using basal medium supplemented with 2 % olive oil, a pH of 6 and a growth temperature of 37 °C. The enzyme extraction process with the addition of 0.25 % Tween 80 increased lipase activity. Implementation of these modifications increased lipase activity by approximately 430 %. The lipase activities produced in the culture supernatant (LCS) and extracted with Tween 80 (LCST80) were characterized. Both extracts hydrolyzed ρ-nitrophenyl (ρNP) esters with different acyl chain lengths, with a preference for short acyl lengths, and had optimum activity at 45 °C. The LCS was stable at acidic and alkaline pH, but LCST80 was only stable at alkaline pH. Methanol, SDS, Triton X-100, EDTA, and EGTA did not affect lipase activity, while divalent cations (Ca²⁺, Zn²⁺, Mg²⁺) - with the exception of Co²⁺— increased lipase activity. Both extracts showed transesterification activity on ρNP ester substrates, and both were able to hydrolyze different natural lipids. The characterization of lipase produced by Luteibacter sp. introduces this recently described genus as a new source of lipases with great biotechnological potential.     

Physiological regulation and optimization of lipase activity in Pseudomonas aeruginosa EF2.

Physiological regulation of extracellular lipase activity by a newly-isolated, thermotolerant strain of Pseudomonas aeruginosa (strain EF2) was investigated by growing the organism under various conditions in batch, fed-batch and continuous culture. Lipase activity, measured as the rate of olive oil (predominantly triolein) hydrolysis, was weakly induced by general carbon and/or energy limitation, strongly induced by a wide range of fatty acyl esters including triglycerides, Spans and Tweens, and repressed by long-chain fatty acids including oleic acid. The highest lipase activities were observed during the stationary phase of batch cultures grown on Tween 80, and with Tween 80-limited fed-batch and continuous cultures grown at low specific growth rates. The lipase activity of Tween 80-limited continuous cultures was optimized with respect to pH and temperature using response surface analysis; maximum activity occurred during growth at pH 6.5, 35.5 degrees C, at a dilution rate of 0.04 h-1. Under these conditions the culture exhibited a lipase activity of 39 LU (mg cells)-1 and a specific rate of lipase production (qLipase) of 1.56 LU (mg cells)-1 h-1 (1 LU equalled 1 mumol fatty acid released min-1). Esterase activity, measured with p-nitrophenyl acetate as substrate, varied approximately in parallel with lipase activity under all growth conditions, suggesting that a single enzyme may catalyse both activities.     

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.     

Human mitochondrial aldehyde dehydrogenase substrate specificity: comparison of esterase with dehydrogenase reaction.

Substrate specificity of human mitochondrial low Km aldehyde dehydrogenase (EC 1.2.1.3) E2 isozyme has been investigated employing p-nitrophenyl esters of acyl groups of two to six carbon atoms and comparing with that of aldehydes of one to eight carbon atoms. The esterase reaction was studied under three conditions: in the absence of coenzyme, in the presence of NAD (1 mM), and in the presence of NADH (160 microM). The maximal velocity of the esterase reaction with p-nitrophenyl acetate and propionate as substrates in the presence of NAD was 3.9-4.7 times faster than that of the dehydrogenase reaction. Under all other conditions the velocities of dehydrogenase and esterase reactions were similar; the lowest kcat was for p-nitrophenyl butyrate in the presence of NAD. Stimulation of esterase activity by coenzymes was confined to esters of short acyl chain length; with longer acyl chain lengths or increased bulkiness (p-nitrophenyl guanidinobenzoate) no effect or even inhibition was observed. Comparison of kinetic constants for esters demonstrates that p-nitrophenyl butyrate is the worst substrate of all esters tested, suggesting that the active site topography is uniquely unfavorable for p-nitrophenyl butyrate. This fact is, however, not reflected in kinetic constants for butyraldehyde, which is a good substrate. The substrate specificity profile as determined by comparison of kcat/Km ratios was found to be quite different for aldehydes and esters. For aldehydes kcat/Km ratios increased with the increase of chain length; with esters under all three conditions, a V-shaped curve was produced with a minimum at p-nitrophenyl butyrate.     

Extracellular carboxylesterase activity of Fusarium graminearum

Summary The extracellular carboxylesterase produced by Fusarium graminearum was investigated. The esterase showed optimum activity at 37°C and at pH 8.7–9.0. Increased activity was observed when whey as a growth substrate was supplemented with either Tween 40 or olive oil. Good activity was also obtained when the fungus was grown on a semi-solid bran culture medium. The esterase showed increased activity towards esters containing short-chain fatty acids.     

Peptidase activity in the inner membrane of Pseudomonas aeruginosa

The location of peptidase activity within the cell envelope structure of Pseudomonas aeruginosa has been studied. Inner and outer membrane fractions were separated on the basis of buoyant density using two consecutive sucrose steps gradients and identified on the basis of known components. The inner membrane was shown to contain peptidase activity while the outer membrane contained none. These data support the hypothesis that P. aeruginosa transports intact peptides.     

Esterase EstE from Xanthomonas vesicatoria ( Xv_EstE) is an outer membrane protein capable of hydrolyzing long-chain polar esters

A new esterase gene from Xanthomonas vesicatoria (formerly X. campestris) DSM 50861 was identified, cloned from a chromosomal gene library and overexpressed in Escherichia coli. The corresponding DNA fragment contains an ORF of 1,818 bp, encoding a hydrolase of the GDSL esterase family. A protein of about 67 kDa, named Xv_EstE, was expressed from this fragment. A N-terminal signal peptide was processed under low-expression conditions, yielding a 63-kDa mature protein. The predicted amino acid sequence showed distinct homology to esterases of the GDSL family. Based on homology, a catalytic triad Gly-Asp-Ser could be defined. Amino acid sequence alignments and computer-assisted structure prediction indicated the presence of a carboxyl-terminal beta-barrel membrane domain which might facilitate binding of Xv_EstE to the outer membrane. This could be verified by differential cell fractionation experiments, in which Xv_EstE was exclusively found in the outer membrane fraction. Xv_EstE showed preferential hydrolytic activity on short chain (up to C(8)) and para-substituted nitrophenylesters as substrates. However, only long-chain 1-hydroxy-pyrene-3,6,8-trisulfonic acid (HPTS)-fatty acid esters were hydrolyzed. Xv_EstE was also found to be active on a series of substrates of industrial interest, such as 1-methylprop-2-ynyl acetate, for which an enantioselectivity up to 93% ee could be recognized.     

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.     

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.     

Studies on catalytic properties of purified high molecular weight pancreatic lipase.

The properties of the 500-fold purified high-molecular-weight lipase have been studied. The rate of hydrolysis of the triglycerides decreases with increasing fatty acid chain length. The lipolytic activity also increases with increase in unsaturation in the fatty acyl moiety. Diglycerides are hydrolyzed at more than twice the rate for triglycerides while monoglycerides are not hydrolyzed. Methyl esters are generally hydrolyzed at a higher rate which increases with increasing chain length of the fatty acid but the enzyme does not act on phospholipids. Emulsifying agents such as Tween 20, gum arabic, and albumin increase the rate of hydrolysis. Metal ions such as Hg2+, Zn2+, Cu2+, and Fe2+ strongly inhibit the lipolytic activity of the high-molecular-weight lipase while Ca2+ or Mg2+ by themselves show no stimulating effect. Treatment of the high-molecular-weight lipase with P-chloromercurybenzoate inhibits hydrolytic activity by 70% while iodoacetic acid has no effect.     

Effects of the detergent Tween 80 on Thermomonospora curvata

Tween 80 (0.1%, v/v) added to Thermomonospora curvata growing in minimal medium caused a transient lowering of the dry cell mass, decreased the optimal growth temperature of the thermophile from 62 to 54°C, and increased extracellular esterase activity. Cells grown in the presence of Tween 80 had decreased concentrations of branched chain fatty acids and increased concentrations of oleic acid. The detergent removed surface protuberances from mycelia and increased the liberation of enzymes active against crystalline cellulose, but did not stimulate liberation of enzymes active against carboxymethylcellulose, starch or pectin.     

Susceptibility of compound 48/80-sensitized Pseudomonas aeruginosa to the hydrophobic biocide triclosan

Pseudomonas aeruginosa is intrinsically resistant to the hydrophobic biocide triclosan, and yet it can be sensitized to low concentrations by permeabilization of the outer membrane using compound 48/80. A selective plating assay revealed that compound 48/80-permeabilized YM64, a triclosan-recognizing efflux pump-deficient variant, was unable to initiate growth on a medium containing triclosan. Macrobroth dilution assay data revealed that treatment with compound 48/80 synergistically decreased minimal inhibitory concentrations of the hydrophobic antibacterial agents rifamycin SV and chloramphenicol for all cell envelope variant strains examined. A low concentration of triclosan exerted a transient bactericidal effect on permeabilized wild-type strain PAO1, after which exponential growth resumed within 4 h. Permeabilized strain YM64 was unable to overcome the inhibition; yet, both strains remained susceptible to chloramphenicol for as long as 6 h, thereby suggesting that the outer membrane remained permeable to nonpolar compounds. These data support the notion that the transitory nature of compound 48/80 sensitization to triclosan in P. aeruginosa does not involve obviation of the hydrophobic diffusion pathway through the outer membrane. The inability of strain YM64 to overcome the synergistic effect of compound 48/80 and triclosan strongly suggests that triclosan-recognizing efflux pumps are involved in maintaining viability in wild-type cells whose outer membranes are otherwise compromised.     

Purification and some properties of a thermostable acid esterase from Acidiphilium sp. AIU 409

Extracellular and cell-bound esterases produced by Acidiphilium sp. AIU 409 were homogeneously purified from culture broth and cells, respectively, and some properties were investigated. Both esterases more rapidly hydrolyzed p-nitrophenyl acyl esters containing long-chain fatty acids from C 8:0 to C 18:0 than those containing short-chain fatty acids from C 2:0 to C 6:0. The Km values for p-nitrophenyl long-chain fatty acid esters from C 8:0 to C 18:0 were approximately 1.3-1.5 mM. The enzymes were stable at 50 degrees C for 2 days between pH 3.0 and 6.5, and optimum pH and temperature were 5.0 and 70 degrees C, respectively. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride and SDS. The molecular mass of both enzymes was estimated to be approximately 64 kDa by SDS-PAGE. The 23 amino acid sequence from the NH(2)-terminus was also the same in both enzymes. These results suggest that extracellular esterase might be composed of the same components as cell-bound esterase.     

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.