In vitro hydrolysis of prostaglandin F2α, esters by serum or plasma of different animal species

The methyl ester of prostaglandin F_2α was hydrolyzed by undiluted human serum with a t1/2 of about 5 min, and the ethyl ester was hydrolyzed at one third of this rate. The 2′-propyl and 3′-pentyl esters were de-esterified at a rapid initial rate and at a slower second rate beginning after 10 min incubation. Alterations at the carbon-15 position of prostaglandin F_2α such as 15(S)-15-methyl or the 15-acetate or 15-hexanoate resulted in a reduction in the rate of hydrolysis of the primary esters at carbon-1.Species variation in serum esterase was very large, with rat serum showing activity more than 500 times that of human serum. Rates of hydrolysis in monkey serum were lower than that of human, and activity in the peripheral blood of the dog was extremely low. Plasma esterase activity in the mesenteric blood of the dog was several times higher than that found in the plasma from the peripheral circulation.     

Specificity and induction of undecyl acetate esterase from Pseudomonas cepacia grown on 2-tridecanone

Undecyl acetate esterase from Pseudomonas cepacia grown on 2-tridecanone was strongly inhibited by organophosphates and other esterase inhibitors. Also, p-chloromercuribenzoate at 1 x 10(-4) M showed a 70% inhibition of esterase activity. The enzyme hydrolyzed both aliphatic and aromatic acetate esters at substrate concentrations of 0.25 M. Under these conditions the highest reaction rate was toward undecyl acetate. No lipase or proteolytic activity was demonstrated. Undecyl acetate esterase was classified as a carboxylesterase (B-esterase). Cell-free activity studies on the production of undecyl acetate esterase grown on different carbon sources plus zymogram studies demonstrated that the enzyme was inducible when 2-tridecanone, 2-tridecanol, undecyl acetate and, to a lesser extent, 1-undecanol were growth substrates. Induction of undecyl acetate esterase during oxidation of 2-tridecanone supports the view that undecyl acetate is an intermediate in the degradation of the methyl ketone.     

ON THE ASSOCIATION OF NON-SPECIFIC ESTERASE ACTIVITY WITH CENTRAL NERVE MYELIN PREPARATIONS

Abstract— Isolated bovine central nerve myelin sheath preparations showed non-specific esterase activity towards naphthyl ester substrates of increasing chain length from acetate to palmitate. Short chain esters were hydrolysed much faster than long chain substrates by myelin, the specific activity for the hydrolysis of β-naphthyl acetate being the highest. Micro-somal fractions from brain white matter were much higher in esterase activity to all naphthyl ester substrates. NADPH-cytochrome c reductase activity was absent from isolated myelin samples. Distilled water and salt and buffer solutions of different ionic strengths and pH were ineffective in releasing non-specific esterase activity from myelin although tri-potassium citrate caused marked inhibition of the membrane-bound esterase activity. The detergent Triton X-100 released esterase activity from the myelin preparations but at a concentration of 0.1 per cent was also inhibitory.     

Fatty acid synthesis is a target for antibacterial activity of unsaturated fatty acids

Long-chain unsaturated fatty acids, such as linoleic acid, show antibacterial activity and are the key ingredients of antimicrobial food additives and some antibacterial herbs. However, the precise mechanism for this antimicrobial activity remains unclear. We found that linoleic acid inhibited bacterial enoyl-acyl carrier protein reductase (FabI), an essential component of bacterial fatty acid synthesis, which has served as a promising target for antibacterial drugs. Additional unsaturated fatty acids including palmitoleic acid, oleic acid, linolenic acid, and arachidonic acid also exhibited the inhibition of FabI. However, neither the saturated form (stearic acid) nor the methyl ester of linoleic acid inhibited FabI. These FabI-inhibitory activities of various fatty acids and their derivatives very well correlated with the inhibition of fatty acid biosynthesis using [(14)C] acetate incorporation assay, and importantly, also correlated with antibacterial activity. Furthermore, the supplementation with exogenous fatty acids reversed the antibacterial effect of linoleic acid, which showing that it target fatty acid synthesis. Our data demonstrate for the first time that the antibacterial action of unsaturated fatty acids is mediated by the inhibition of fatty acid synthesis.     

Human liver aldehyde dehydrogenase. Esterase activity.

Human liver aldehyde dehydrogenase has been found to be capable of hydrolyzing p-nitrophenyl esters. Esterase and dehydrogenase activities exhibited identical ion exchange and affinity properties, indicating that the same protein catalyzes both reactions. Competitive inhibition of esterase activity by glyceraldehyde and chloral hydrate furnished evidence that p-nitrophenyl acetate was hydrolyzed at the aldehyde binding site for dehydrogenase activity. Pyridine nucleotides modified esterase activity; NAD+ accelerated the rate of p-nitrophenyl acetate hydrolysis more that 5-fold, whereas NADH increased activity by a factor of 2. Activation constants of 117 muM for NAD+ and 3.5 muM for NADH were obtained from double reciprocal plots of initial rates as a function of modifier concentration at pH 7. The kinetics of activation of ester hydrolysis were consistent with random addition of pyridine nucleotide modifier and ester substrate to this enzyme.     

Further Studies on a Serum Nonantibody α Globulin with Antistaphylococcal Activity

S ummary . Previous work in this laboratory indicated that normal human serum contains a pseudoglobulin which inhibited the growth and respiration of staphylococci. Studies with a variety of serum fractions suggest that antibacterial agent (ABA) and α globulin alone possess the antibacterial properties which distinguish them from all other fractions tested. ABA has been prepared from unfiltered and Seitz filtered (to remove the β lysin) sera, platelet-rich plasmas of humans, rabbits, guineapigs and rats and assayed by a sensitive rapid radioisotopic technique. ABA is widely distributed among animal sera and, in contrast to the human ABA, requires a 2–4 fold increase in the concentration of coagulase necessary to demonstrate a comparable reversal of the antirespiratory effect. Immunological studies confirmed the nonantibody nature of ABA. ABA did not react with antisera to IgA, IgG, IgM, human β lipoprotein, haptoglobin, thyroglobulin, C-reactive protein, ceruloplasmin, fibrinogen or antiserum to β lysin obtained from other workers. Of the various serum fractions tested only á globulin had any detectable antistaphylococcal activity.     

Estradiol esters can replace 17 beta-estradiol in the stimulation of DNA and esterase synthesis by MCF-7 cells: a possible role for the estrogen-sensitive MCF-7 cell esterase

In this communication we extend our earlier observations on estrogen-sensitive carboxyl esterases in MCF-7 human breast cancer cells able to hydrolyze esters of estradiol. Using either estradiol acetate or p-nitrophenyl hexanoate as substrates, esterase activity was found to increase 2-3-fold in MCF-7 cells maintained in the presence of 10(-8) M estradiol. Following sucrose density centrifugation, over 85% of total esterase activity was found in the cytoplasmic fraction. No esterase activity was found in spent media from growing cells. By size exclusion chromatography, estradiol acetate esterase activity exhibited a mol. wt of 45-50 kDa. Attempts to demonstrate incorporation of [3H]estradiol into estradiol fatty acid esters by the above MCF-7 cell line (203P) were unsuccessful, although, such incorporation could be demonstrated in two other MCF-7 cell sublines. Incubation of the 203P cells with 10 nM [3H]estradiol in the presence of 0.5 mM radioinert estradiol acetate resulted in the incorporation of 35 +/- 12% of the label into the estradiol acetate in 10 min. In the absence of radioinert estradiol acetate, no incorporation was observed. When MCF-7 cells were incubated with [3H]estradiol in the presence of a large excess of radioinert estradiol valerate, label was found only in estradiol valerate. Similarly, when the incubation was carried out in the presence of a mixture of radioinert estradiol acetate and valerate, label was incorporated into both esters. We conclude that the apparent formation of radiolabeled estradiol esters by MCF-7 cells incubated under the above conditions, results at least in part, from an esterase-catalyzed exchange reaction. Under conditions where no ester hydrolysis could be detected in the absence of cells, valerate and stearate esters of estradiol were found to be as effective as unesterified estradiol in stimulating esterase synthesis and the incorporation of [3H]thymidine into DNA. These results are consistent with a model in which an intracellular esterase in MCF-7 cells can generate estradiol from an exogenous lipoidal steroid and elicit an estrogen response.     

The requirement for esterase activation in the anti-immunoglobulin-triggered movement of B lymphocytes.

In this paper we bring evidence suggesting that there is activation of an esterase upon reaction of anti-immunoglobulin antibodies (anti-Ig) with murine B lymphocytes. B lymphocytes upon exposure to anti-Ig cap the ligand-receptor complexes and immediately afterward become briefly motile. It is this latter step which is inhibitable by exposure to di-isopropyl phosphofluoridate (DFP). Various experimental manipulations indicated that treatment with anti-Ig activates the cell for motility which, however, is not manifested until the temperature is raised to 37 degrees C. The cell incubated with anti-Ig at cold temperatures becomes susceptible to the effect of DFP, suggesting that the antibody-treated cells are activated up to but not beyond the DFP inhibitable step. Exposure of cells to DFP and removal of it before their treatment with anti-Ig does not affect the anti-Ig-induced response. Four lines of evidence indicate that the reduction of lymphocyte movement of DFP is due to the inhibition of an esterase activated by the combination of antibody and cell: 1) The inhibition by DFP is irreversible; once DFP has reacted it can be washed away and the antibody-treated cell is still inhibited. 2) The inhibition increases with time of contact of lymphocytes and DFP and with the concentration of DFP. 3) A very poorly phosphorylating phosphonate, phenyl ethyl pentylphosphonate is completely inactive under conditions where an excellent phosphorylating phosphonate, p nitrophenyl ethyl pentylphosphonate maximally inactivated the cells' movement. 4) The amino acid esters, tosyl L arginine methyl ester and benzoyl arginine methyl ester specifically prevent the inactivation by DFP. The last finding suggests that tosyl L arginine methyl ester and benzoyl L arginine methyl ester might be substrates for the putative antibody-induced lymphocyte esterase. Lymphocytes incubated with antibody in the cold for more than 30 min lose their ability to move when the temperature is raised, suggesting that there is a time-dependent deactivation of the cell.     

The enzymatic nature of human c1r: a subcomponent of the first component of complement.

The esterase activity of the C1r subcomponent of the first component of complement has been investigated. C1r was found to hydrolyze two amino acid methyl esters; N-acetyl-L-arginine methyl ester and N-acetyl-glycyl-L-lysine methyl ester, and two amino acid p-nitrophenyl esters, N-carbobenzyloxy-L-tyrosine-p-nitrophenyl ester and N alpha-carbobenzyloxy-L-lysine-p-nitrophenyl ester. A detailed kinetic analysis of the hydrolysis of N-Z-L-Tyr-ONp by C1r revealed that the enzymatic activity per microgram of protein decreased as the C1r concentration was increased. The loss of activity suggested that above 0.5 micron C1r was undergoing aggregation with a loss of active sites. Similarly, when C1r was titrated with the active site titrant p-nitrophenyl-P'-guanidinobenzoate the number of titratable sites per milligram of protein decreased with increasing protein concentration. The hydrolysis of N-Z-L-Tyr-ONp by C1r was inhibited by several synthetic inhibitors including phenylmethanesulfonylfluoride, p-amidinophenylmethanesulfonylfluoride, diisopropylfluorophosphate, and p-tosyl-L-lysine-chloromethyl ketone. However, the peptide esterase inhibitors Trasylol, hirudin, leupeptin, and C1 esterase inhibitor had no effect on the esterase activity of C1r.     

Isolation and characterization of sialate 9(4)-O-acetylesterase from influenza C virus

An esterase was isolated from influenza C virus with a specific activity from 1.7-5 U/mg protein, and its substrate specificity was tested with various naturally occurring O-acylated sialic acids, synthetic carbohydrate acetates, and other esters. The enzyme hydrolyses only acetic acid esters at significant rates. The non-natural substrates 4-methyl-umbelliferyl acetate, 4-nitrophenyl acetate, and alpha-naphthyl acetate are cleaved at highest hydrolysis rates, followed by the natural substrate N-acetyl-9-O-acetylneuraminic acid. The esterase also acts on N-glycoloyl-9-O-acetylneuraminic acid and, much slower, on N-acetyl-4-O-acetylneuraminic acid; N-acetyl-7-O-acetylneuraminic acid is not hydrolysed. 2-Deoxy-2,3-didehydro-N-acetyl-9-O-acetylneuraminic acid is also a substrate for this enzyme, however, 6-O-acetylated N-acetylmannosamine and glucose are not. Esterification of the carboxyl function of sialic acids strongly reduces or prevents esterase action on O-acetyl groups. The carboxyl ester is not hydrolysed. The relative cleavage rates also depend on the type of the non-sialic acid part of the molecule. N-Acetyl-9-O-acetylneuraminic acid as component of sialyllactose and rat serum glycoprotein shows hydrolysis rates close to the free form of this sugar, while acetyl ester groups of bovine submandibular gland mucin and rat erythrocytes are hydrolysed at slower rates. Gangliosides and 4-O-acetylated glycoproteins are no substrates for the purified enzyme. A slow hydrolysis is observed by incubation of 9-O-acetylated GD1a with intact influenza C viruses. As other natural acetyl esters (acetyl-CoA and acetylthiocholine iodide) are not hydrolysed, the enzyme can be classified as sialate 9(4)-O-acetylesterase (EC 3.1.1.53).     

Bacteriostatic activity of serum against staphylococci

Cybulska, Janina (State Institute of Hygiene, Warsaw, Poland), and J. Jeljaszewicz. Bacteriostatic activity of serum against staphylococci. J. Bacteriol. 91:953-962. 1966.-Antistaphylococcal activity of normal serum against strains exhibiting various patterns of coagulase, clumping-factor, and staphylokinase production is not connected with the presence of these factors. Purified coagulase does not influence this property of serum. Coagulase-negative strains with clumping-factor activity grow in normal serum as typical pathogenic staphylococci. Serum bacteriostatic activity against staphylococci may be reversed by several nonspecific factors, such as sterile broth, supernatant fluids of coagulase-negative strains, and ammonium sulfate precipitates of culture supernatant fluids of various staphylococci. Immune sera with a high agglutinating titer for staphylococcal cells do not prevent growth of serum-resistant strains; serum-susceptible strains are inhibited as in normal serum control. Activation or blocking of the serum fibrinolytic system does not influence serum bacteriostatic activity. The growth rate of serum-resistant strains is identical in serum and in Todd-Hewitt broth; serum-susceptible strains are inhibited to the inoculum level, but decreases and increases in viable count are noted during a 24-hr observation period. Observations made with sera of 10 animal species clearly demonstrated differences in serum bacteriostatic activity, mouse serum being completely noninhibitory and cat serum only weakly inhibitory. The technique of quantitative determination of serum susceptibility of staphylococci is described, and the importance of serum antistaphylococcal activity in vitro is discussed. Experimental staphylococcal infection produced in rabbits by intravenous injection of different Staphylococcus aureus strains did not result in significant changes in serum antistaphylococcal activity. The technique of experimental infection used caused chronic infection, with a peak on the 14th day; this was proved by means of a newly developed 5'-nucleotidase test. At the same time, sera of infected animals exhibited slight inhibitory properties, which returned to initial values 1 week later. Infection was produced by strains recognized as nonpathogenic and was inhibited in vitro by sera from both normal and infected rabbits. It is concluded that antistaphylococcal activity of serum should be considered as an "in vitro" phenomenon, which seems to have no importance in defense mechanisms of rabbits infected intravenously with staphylococci.     

The 44-kDa c-type cytochrome induced in Rhodobacter capsulatus during growth with dimethylsulphoxide as an electron acceptor is a cytochrome c peroxidase

Abstract The propionate (Pro), decanoate (Dec) and laurate (Lau) esters of 5-(4-hydroxy-3,5-dimethoxyphenylmethylene)-2-thioxothiazoline-3-acetic acid were assessed as substrates for lipase and esterase. On hydrolysis these substrates yield an intensely red coloured phenol which could be assayed at 505 nm. The Pro ester was an effective substrate for porcine esterase and was hydrolysed at a rate 20 times greater than the Lau and Dec esters. Conversely, Pseudomonas Lipase had a high activity towards the Lau and Dec esters, especially in the presence of bovine serum albumin, but little activity towards the Pro ester. The Dec and Lau were used to detect lipolytic activity in Pseudomonas strains associated with milk spoilage. For this purpose, the substrates were absorbed onto filter paper disks, which were placed over bacterial colonies growing on agar plates; activity was indicated by bright red colouration of discs within 2 h. Escherichia coli colonies hydrolysed the Pro but not the Lau or Dec esters.     

Purification and characterisation of an ester hydrolase from a strain of Arthrobacter species: its application in asymmetrisation of 2-benzyl-1,3-propanediol acylates

An ester hydrolase (ABL) has been isolated from a strain of Arthrobacter species (RRLJ-1/95) maintained in the culture collection of this laboratory. The purified enzyme has a specific activity of 1700 U/mg protein and is found to be composed of a single subunit (Mr 32,000), exhibiting both lipase and esterase activities shown by hydrolysis of triglycerides and p-nitrophenyl acetate respectively. Potential application of the enzyme concerns the asymmetrisation of prochiral 2-benzyl-1,3-propanediol esters besides enantioselective hydrolysis of alkyl esters of unsubstituted and substituted 1-phenyl ethanols.     

Biochemical characterisation of esterases active in hydrolysing xenobiotics in wheat and competing weeds

Esterase activities toward model xenobiotic substrates ( p -nitrophenyl acetate, naphthyl acetate) and pesticide esters (diclofop methyl, bromoxynil octanoate, binapacryl) have been compared in crude extracts from wheat (Triticum aestivum L.) and Triticum progenitors of wheat. Esterase activities were also determined in the weeds, wild oat ( Avena fatua ) and two populations of black-grass ( Alopecurus myosuroides ), one of which (Rothamsted) is susceptible to herbicides, while the other (Peldon) shows cross-resistance to multiple classes of herbicides. Esterase activity toward the model substrates was highest in wheat, while the weeds were more active in hydrolysing the pesticides. Using isoelectric focussing (pH 4–8), 13 proteins with esterase activity toward α -naphthyl acetate could be resolved in hexaploid wheat (genome AABBDD). The pattern of these activities was most similar to that of the diploid progenitor T. tauschii (DD), excepting a major acidic esterase (pI 4.6), which originated from T. urartu (AA). Resolved esterase activities in the weeds were distinct from those observed in the Tritcum species. However, unlike the case with other classes of xenobiotic-metabolising enzymes, the complement of esterases in the Peldon and Rothamsted populations of black-grass appeared to be identical. In all species, the more basic esterases (>pI 5.0) were sensitive to inhibition by organophosphate and carbamate insecticides, suggesting that they were B-class esterases. In contrast, the acidic wheat esterase (pI 4.6) with the greatest activity toward α -naphthyl acetate was insensitive to insecticides. This wheat-specific esterase was purified 7000-fold by a combination of hydrophobic interaction chromatography, gel filtration and anion-exchange chromatography. The purified esterase behaved as a monomeric 45-kDa protein showing high activity toward p -nitrophenyl acetate and α -naphthyl acetate, but limited activity toward the pesticides.     

Hydrolysis of dolichyl esters by rat liver lysosomes

Dolichyl ester hydrolase activity is broadly distributed among the organs of the rat. The highest activity was found in spleen, brain, lung, and thyroid tissues, whereas this activity is very low in stomach and intestine. The esterase involved is localized to the lumen of lysosomes and, to some extent, in the plasma membranes. Hydrolysis occurs with both alpha-saturated and alpha-unsaturated polyisoprenes esterified with different fatty acids, but the rate of hydrolysis is strongly dependent on the nature of the substrate. The enzyme involved is inhibited by divalent cations, EDTA and EGTA and also by one of the products, dolichol. The esterase is activated by 3-[(3-cholamidopropyl) dimethylammonio]-1-propranesulfonic acid and taurodeoxycholate and inhibited by Triton X-100. Dolichyl esterase activity is completely inhibited by alpha- and beta-naphthyl acetate, phenylmethylsulfonyl fluoride, and beta-chloromethylmercurisulfate. These inhibitors, as well as the pH optimum for dolichyl ester hydrolysis, clearly differentiate the enzyme involved from cholesteryl esterase and triglyceride lipase. Microsomal phospholipase A hydrolyzes dolichyl esters at a slow rate only. In vivo labeling experiments with [3H]mevalonate demonstrated that newly synthesized dolichol is transported in esterified form to the lysosomes, where this lipid is slowly hydrolyzed by the esterase. The possibility is raised that the role of the fatty acyl moiety may be to target dolichol to its final location in the cell.     

Macrophage esterase: identification, purification and properties of a chymotrypsin-like esterase from lung that hydrolyses and transfers nonpolar amino acid esters.

A chymotrypsin-like esterase was purified from beef lung. This lysosomal enzyme, not previously characterized, seemed to be composed of two or more forms with molecular weights of about 52 000. It hydrolysed N-benzoyl-DL-phenylalanine beta-naphthol ester at acid and neutral pH; it polymerized L-phenylalanine methyl ester(Phe-OMe) at neutral pH; and it transferred the Phe-residue from Phe-OMe to hydroxylamine at neutral pH. Phenylmethanesulfonyl fluoride, an inhibitor of hydrolytic enzymes with serine in their catalytic site, inhibited this enzyme, but pepstatin, the cathepsin D (EC 3.4.4.23) inhibitor, did not. Sulfhydryl reagents were not required for activity. Macrophages, especially pulmonary alveolar macrophages, were a rich source of this esterase, so it is likely that the enzyme purified from lung came from its macrophages. The esterase hydrolysed and transferred monoamino acid esters, especially those of the aromatic type. Cathepsin C, the dipeptidyl peptide hydrolase (EC 3.4.14.1), acted only on dipeptide esters and amides. Pancreatic chymotrypsin acted on both monoamino acid and dipeptide esters. The chymotrypsin-like esterase did not hydrolyse hemoglobin, casein, or plasma albumin. Thus its proteolytic activity, if present, must be limited to specific substrates, as yet unknown.     

Purification and characterization of rat urinary esterase A1

An enzyme, esterase A1, which hydrolyzes tosyl-arginine methyl ester (Tos-Arg-OMe) was separated from esterase A2 and kallikrein of male rat urine and purified by a procedure involving ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography and gel filtration. The resulting preparation was apparently homogeneous, as assessed by polyacrylamide gel electrophoresis. The molecular weight of the preparation was estimated to be 27,000 by SDS-polyacrylamide gel electrophoresis and 30,000 by gel filtration. The enzyme was more specific for arginine methyl esters than for lysine methyl esters. The optimum pH determined with Tos-Arg-OMe as a substrate was 8.0 and the Km was 11.8 mM. The Tos-Arg-OMe esterolytic activity of esterase A1 was inhibited by soybean trypsin inhibitor, but not by aprotinin. In immunodiffusion analysis, the antiserum to esterase A1 formed immunoprecipitin arcs with this enzyme and the urine collected from rat bladder, but not with esterase A2, kallikrein, plasma and the urine collected from ureters. These results indicate that rat urinary esterase A1 differs from esterase A2 and kallikrein. The esterase A1 appears to be produced by accessory sex glands and excreted via the spermiduct into the urine.     

Technical considerations in evaluating the adhesion of leukocytes to aortic endothelium of the rat

Comments on techniques for characterizing leukocytes adhered to the aortic endothelium of the rat are given. Alpha-naphthyl acetate esterase positive leukocytes were studied by optical microscopy of en face intima-media preparations. Results indicate 1) 1% paraformaldehyde-2% glutaraldehyde is a better fixative than formalin-calcium or 4% paraformaldehyde with or without 1.5 mM CaCl2; the latter produces distortion of leukocytes, endothelial desquamation and enzymate inhibition, 2) washing the aorta with phosphate-buffered saline for 90 sec prior to fixation-perfusion produces a notable decrease in the number of leukocytes adhered, 3) diazotized parasaniline is better than fast blue RR salt as coupling agent in the esterase reaction, and 4) counterstaining with 1% methyl green for 1 min, before or after the esterase reaction, is not adequate because of limited contrast and the heavy staining of smooth muscle. Counterstaining with Gill's hematoxylin No. 3 for 90 sec is adequate only when done before the esterase reaction. Inhibition of endothelial esterase activity by hematoxylin decreases background, favors contrast of adhered leukocytes and makes it possible to observe nucleus-cytoplasm relations.     

Preliminary characterisation of esterase and platelet-activating factor (PAF)-acetylhydrolase activities from cat flea (Ctenocephalides felis) salivary glands

Naphthyl esterase and platelet-activating factor (PAF)-acetylhydrolase activities were detected in the salivary glands of the cat flea, Ctenocephalides felis. Salivary naphthyl esterase activity is disgorged during exploratory probing. Whole extracts of salivary glands contain esterase activity against the short-chain naphthyl esters alpha-naphthyl acetate (approximately 210pmol/min/gland pair; 10.0micromol/min/mg specific activity; K(m) approximately 59microM) and beta-naphthyl acetate (approximately 110pmol/min/gland pair; 5.2micromol/min/mg specific activity; K(m) approximately 132microM). Salivary gland extracts have PAF-acetylhydrolase activity (approximately 5pmol/min/gland pair; 0.24micromol/min/mg specific activity) but do not have detectable acetylcholinesterase activity. Native-PAGE and IEF resolve three and six salivary gland naphthyl esterase bands, respectively, and both patterns are different from carcass esterases. Salivary gland naphthyl esterase activity binds reversibly to Concanavalin A, and enzymatic deglycosylation with glycopeptidase F produced a new, fast-migrating salivary gland naphthyl esterase band on Native-PAGE. Renaturation of esterase activity after SDS-PAGE gave approximately 56kDa, approximately 57kDa and approximately 58kDa naphthyl-esterase-positive bands. On gel filtration naphthyl esterase and PAF-acetylhydrolase activities co-elute as a single peak with an apparent molecular weight of approximately 59kDa. This partially purified pool of enzyme had esterase activity against a series of short-chain alpha- and beta-naphthyl esters. The heterogeneity of salivary gland esterases, their relationship to PAF-acetylhydrolase, and the possible physiological functions of salivary gland PAF-acetylhydrolase activity are discussed.     

Decreasing the level of ethyl acetate in ethanolic fermentation broths of Escherichia coli KO11 by expression of Pseudomonas putida estZ esterase

During the fermentation of sugars to ethanol relatively high levels of an undesirable coproduct, ethyl acetate, are also produced. With ethanologenic Escherichia coli strain KO11 as the biocatalyst, the level of ethyl acetate in beer containing 4.8% ethanol was 192 mg liter(-1). Although the E. coli genome encodes several proteins with esterase activity, neither wild-type strains nor KO11 contained significant ethyl acetate esterase activity. A simple method was developed to rapidly screen bacterial colonies for the presence of esterases which hydrolyze ethyl acetate based on pH change. This method allowed identification of Pseudomonas putida NRRL B-18435 as a source of this activity and the cloning of a new esterase gene, estZ. Recombinant EstZ esterase was purified to near homogeneity and characterized. It belongs to family IV of lipolytic enzymes and contains the conserved catalytic triad of serine, aspartic acid, and histidine. As expected, this serine esterase was inhibited by phenylmethylsulfonyl fluoride and the histidine reagent diethylpyrocarbonate. The native and subunit molecular weights of the recombinant protein were 36,000, indicating that the enzyme exists as a monomer. By using alpha-naphthyl acetate as a model substrate, optimal activity was observed at pH 7.5 and 40 degrees C. The Km and Vmax for alpha-naphthyl acetate were 18 microM and 48.1 micromol. min(-1). mg of protein(-1), respectively. Among the aliphatic esters tested, the highest activity was obtained with propyl acetate (96 micromol. min(-1). mg of protein(-1)), followed by ethyl acetate (66 micromol. min(-1). mg of protein(-1)). Expression of estZ in E. coli KO11 reduced the concentration of ethyl acetate in fermentation broth (4.8% ethanol) to less than 20 mg liter(-1).