Β-naphthyl Esters Research Articles

Purification of an organic solvent-tolerant lipase from Aspergillus niger MYA 135 and its application in ester synthesis

Abstract An organic solvent-tolerant lipase from olive oil-induced Aspergillus niger MYA 135 supernatant was purified using two methods: electroelution and ion-exchange chromatography. With electroelution purification was 8.4-fold and recovery 47% and with ion-exchange 16.6-fold and 53.4%, respectively. The purified enzyme showed a prominent single band with SDS-PAGE and was a monomeric protein of 68 kDa. The isoelectric point (pI) of the lipase was 5.1 and optimum pH and temperature for activity were 7.0 and 37 °C, respectively. The lipase showed affinity for esters with long acyl chains, with a K m of 0.99 mM for C18. Substrate specificity of the immobilized lipase was highest for C18 among the various α- and β-naphthyl esters assayed. Substrate specificity agreed with kinetics parameters of long-chain fatty acids (C18). Transesterification activity of the A. niger MYA 135 lipase indicates that it could be a potential biocatalyst for biodiesel production.

The SGNH-hydrolase of Streptomyces coelicolor has (aryl)esterase and a true lipase activity

The Streptomyces coelicolor A3(2) gene SCI11.14c was overexpressed and purified as a His-tagged protein from heterologous host, Streptomyces lividans. The purification procedure resulted in 34.1-fold increase in specific activity with an overall yield of 21.4%. Biochemical and physical properties of the purified enzyme were investigated and it was shown that it possesses (aryl)esterase and a true lipase activity. The enzyme was able to hydrolyze p-nitrophenyl-, α- and β-naphthyl esters and poly(oxyethylene) sorbitan monoesters (Tween 20–80). It showed pronounced activity towards p-nitrophenyl and α- and β-naphthyl esters of C 12–C 16. Higher activity was observed with α-naphthyl esters. The enzyme hydrolyzed triolein (specific activity: 91.9 U/mg) and a wide range of oils with a preference for those having higher content of linoleic or oleic acid (C18:2; C18:1, cis). The active-site serine specific inhibitor 3,4-dichloroisocoumarin (DCI) strongly inhibited the enzyme, while tetrahydrofurane and 1,4-dioxane significantly increased (2- and 4- fold, respectively) hydrolytic activity of lipase towards p-nitrophenyl caprylate. The enzyme exhibited relatively high temperature optimum (55 °C) and thermal stability. CD analysis revealed predominance of α-helical structure (54% α-helix, 21% β-sheet) and a T m value at 66 °C. Systematic bioinformatic analysis of deduced amino acid sequence of S. coelicolor enzyme placed it to the SGNH-hydrolase family. Phylogenetic analysis of the predicted protein homologous to the S. coelicolor SGNH-hydrolase generated three distinct groups consisting of proteins from Actinomycetales, Ascomycota and Nematoda. At present it seems that these enzymes are most conserved among soil inhabiting organisms.

Molecular Characterization of a Novel Family VIII Esterase from Burkholderia multivorans UWC10

An esterase producing Burkholderia multivorans UWC10 strain was isolated by culture enrichment. A shotgun library of B. multivorans UWC10 genomic DNA was screened for esterase activity and a recombinant clone conferring an esterolytic phenotype was identified. Full-length sequencing of the DNA insert showed that it consisted of a single open reading frame (ORF1) encoding a predicted protein of 398 amino acids. ORF1 (termed EstBL) had a high protein sequence identity to family VIII esterases. The EstBL primary structure showed two putative serine motifs, G-V-S₁₄₉-D-G and S₇₄-V-T-K. The estBL gene was successfully over-expressed in E. coli and the encoded protein purified by a combination of ammonium sulphate fractionation, hydrophobic interaction, ion exchange and size exclusion chromatographies. Biochemical assays confirmed EstBL esterase activity and revealed a preference for short-chain p-nitrophenyl and β-naphthyl esters (C2-C4) with no activity against β-lactam substrates. Secondary structure predictions indicated that EstBL adopts the α/β fold, which is common to all esterases.

Differentiation of lactic acid bacteria strains by postelectrophoretic detection of esterases.

Lactic acid bacteria (LAB) comprise a diverse group of Gram-positive, non-spore-forming microorganisms. These bacteria are widely used in food technology. The species identification of LAB depends mainly on physiological and biochemical criteria. The esterolytic systems of LAB remain poorly characterized. Esterases (EC 3.1.1.3) represent a diverse group of hydrolases catalyzing the cleavage and formation of esters bonds Screening of esterases is usually performed either by employing chromophoric substances (e.g., alpha- or beta-naphthyl esters of short-chain fatty acids). The post-electrophoretic detection of esterases is a sensitive technique applied in bacterial systems, that mainly provides information on the similarity of strains within the same species or subspecies according to their esterase patterns. This technique is principally used to determine the number and substrate specificity of esterases and lipases, revealing the complexity of lipase and esterase systems. The present chapter describes the technique of polyacrylamide gel electrophoresis (PAGE; in the absence of sodium dodecyl sulfate [SDS]), in non-denaturing conditions, to find intracellular fractions for strain typing of LAB.

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 α-naphthyl acetate (∼210 pmol/min/gland pair; 10.0 μmol/min/mg specific activity; K m∼59 μM) and β-naphthyl acetate (∼110 pmol/min/gland pair; 5.2 μmol/min/mg specific activity; K m∼132 μM). Salivary gland extracts have PAF-acetylhydrolase activity (∼5 pmol/min/gland pair; 0.24 μmol/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 ∼56 kDa, ∼57 kDa and ∼58 kDa 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 ∼59 kDa. This partially purified pool of enzyme had esterase activity against a series of short-chain α- and β-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.

Distribution and Inhibition of Esterases in Various Body Tissues of Susceptible and Resistant German Cockroaches (Dictyoptera: Blattellidae)

The distribution and inhibition of esterase activity among body of the resistant Crawford and susceptible CSMA strains of male German cockroach, Blattella germanica (L.), were evaluated with α- and β-naphthyl esters using a spectrophotometric assay and native polyacrylamide gel electrophoresis. The esterase activities in the Crawford strain were significantly higher than in the CSMA strain in most tissues except midgut and male genitalia, where the activities were similar. In both strains, isozyme a was dominant in the brain and fat body and isozymes c and d were more abundant in the midgut; but in addition to isozyme a, isozyme b was observed only in the Crawford strain. The α-naphthyl butyrate (α-NB) and β-naphthyl acetate (β-NA) esterase activities in the brain of both strains were completely inhibited by most inhibitors (DEF, 3,4-dichloroisocoumarine, paraoxon, pepstatin A, and propoxur). However, in the midgut tissue and contents, the activities and inhibition patterns of the α-NB and β-NA esterase were apparently different from the brain and between strains. The complete inhibition by paraoxon was observed regardless of the strain, substrate type, and body part for enzyme source. In esterase inhibition on native PAGE gels, paraoxon completely inhibited the activity of most esterases similar to the results from spectrophotometric assay. In addition, the esterase b was observed from the whole body homogenates of several German cockroach resistant strains on native PAGE analysis.

Isolation and Characterization of a Tributyrin Esterase from Lactobacillus plantarum 2739

An intracellular tributyrin esterase from Lactobacillus plantarum 2739 was purified to homogeneity by chromatography on DEAE cellulose, Sephacryl 200, carboxymethylcellulose, and Mono Q. The enzyme E2 was separated on DEAE cellulose from a second esterase, E1, and a minor esterase. Additional minor esterases were separated from E2 during chromatography on Sephacryl. E2 was a monomer with a relative molecular mass of approximately 85 kDa. The enzyme was most active at pH 7 and 35°C and retained about 30% of maximal activity at pH 5 and about 18% at 12°C. E2 was strongly inhibited by 1mM phenylmethylsulfonyl fluoride, Hg2+, or Ag+ and was moderately stimulated by Ca2+ and Mg2+. E2 was active on β-naphthyl esters of fatty acids from C2 to C10 with a preference for β-naphthyl butyrate. Tributyrin and, to a lesser extent, tricaprylin and milk fat were also hydrolyzed. Partially purified E1 was more active on tributyrin than was E2. The sequence of the first 15 N-terminal amino acids of purified E2 was Ser-Asn-Glu-His-Thr-Gln-Glu-Val-Leu-Asn-Gln-Thr-Val-Ala-Asp. The enzyme showed a decimal reduction value at 70°C of 2.5min. The Michaelis constant of E2 on β-naphthyl butyrate was 0.36mM.

Purification and characterization of an intracellular esterase from Brevibacterium linens ATCC 9174

An intracellular esterase from Brevibacterium linens ATCC 9174 was purified 44-fold to homogeneity using ammonium sulphate fractionation, anion exchange chromatography, hydrophobic interaction chromatography and anion exchange chromatography. The pH and temperature optima were 7.5 and 35 °C, respectively. The molecular mass of the enzyme was found to be 54 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis and 201 kDa by gel filtration, indicating that the native enzyme exists as a tetramer. The pI of the purified esterase was 4.0. The esterase was activated by ethylenediaminetetraacetic acid, but strongly inhibited by N-ethylmaleimide and p-hydroxymercuribenzoate, Cd2+, Zn2+ and Hg2+; activity was increased by dithiothreitol and cysteine at 0.1 mM, but decreased at 1.0 mM. The esterase hydrolysed β-naphthyl esters of acetic, butyric, caproic, caprylic and capric acids, but not of lauric, myristic, palmitic or oleic acids. Km and Kcat values for β-naphthyl butyrate were 0.1 mM and 0.2 s−1, respectively, while the corresponding values for β-naphthyl caproate were 0.03 mM and 0.5 s−1, respectively. The sequence of the first 19 N-terminal amino acids was NH2-Ala-Val-Tyr-Ser-Ala-Pro-Gly-Thr-Glu-Gly-Ser-Leu-Val-Thr-Phe-Lys-Pro-Arg-Tyr.

Isolation and characterization of an extracellular lipase from Pseudomonas tolaasii

Pseudomonas tolaasii, a plant pathogen, produced an extracellular, heat-stable lipase in reconstituted skim milk (10%, w v ) which was purified by ion-exchange chromatography on DEAE-52 cellulose and gel filtration on Sephadex G-150. The purified enzyme showed one major protein peak on a TSK G3000 SW column, from which a native molecular weight of ~670 kDa was estimated. Maximum enzyme activity was at pH 7.0 and 35 °C when assayed on β-naphthyl caprylate. The enzyme lost no activity at pH 7.0 for 48 h at 21 °C and was more stable over the pH range 5–6.5 than in the range 8–11. The lipase was quite heat-stable (D-values ranged from 1171 s at 100 °C to 79 s at 140 °C; the Z-value in the temperature range 100–140 °C was ~31 °C and the corresponding activation energy (E a) for inactivation was 94 kJ mol −1. The enzyme was inhibited more strongly by EDTA than by o-phenanthroline but not by phenylmethylsulphonylfluoride (PMSF) and was strongly reactivated by Ca 2+. The lipase hydrolysed β-naphthyl esters in the order C 8 > C 6 > C 4 > C 2 > C 10.

Purification and characterization of a cell surface-associated esterase from Lactobacillus fermentum DT41

A cell surface-associated esterase from Lactobacillus fermentum DT41, a starter used to produce Parmesan cheese, was purified to homogeneity by chromatography on Q-Sepharose, Sephacryl 200, Phenyl-Superose and Mono Q. The enzyme, together with a distinct cytoplasmic esterase, expressed the highest activity during the exponential phase of growth. The esterase was a monomer with a M r of ca 67 kDa and was most active at pH 7.0 and 30–35 °C, retaining considerable activity at pH 5.0 and 15 °C. The enzyme was stable at the cooking temperature (54–56 °C for a few minutes) of Parmesan cheese, its D 55 °C value was 35 min. The highest activity was determined on β-naphthyl butyrate, but marked hydrolysis also occurred with β-naphthyl esters of C2 to C10 fatty acids. β-Naphthyl esters of C14 to C18:1 fatty acids were not hydrolyzed and only tributyrin was degraded among the triglycerides. The K m on β-naphthyl butyrate was 0.31 mM with a V max of 140 μmol min −1 mg −1 . The esterase was strongly inhibited by 5 mM phenylmethylsulfonyl fluoride and by 1 mM Hg 2+ and Ag + , and moderately stimulated by Ca 2+ and Mg 2+ .

PURIFICATION AND CHARACTERIZATION OF A LIPASE FROM LACTOBACILLUS PLANTARUM 2739

A 65 kDa intracellular lipase from Lactobacillus plantarum 2739 was purified to homogeneity (482-fold, specific activity of 251 μmol/mg per min) and characterized. The purification procedure included chromatography with Q-Sepharose, Sephacryl 200, Phenyl-Superose and Mono Q. The purified lipase was optimally active at pH 7.5 and 35C; it retained about 40% of the maximum activity at pH 5.0 and 15C. The enzyme was stable at 65C (D65C= 18.6 min) and was irreversibly inactivated at 75C for 2 min. On triglycerides, the highest activity was determined on tributyrin but trilaurin and tripalmitin were hydrolyzed also. The Km on tributyrin was 2.31 mM. β-Naphthyl esters of fatty acids from C2 to C12 were hydrolyzed with a preference for β-naphthyl butyrate. After lipolysis, the fatty acid profiles in β-monoacylglycerols of milk fat showed similarities among porcine pancreatic lipase, rennet paste and lipase from Lb. plantarum 2739, but the bacterial enzyme caused a greater hydrolysis of C10 and C12 fatty acids esterified at the Sn-2 position of glycerol. The lipase was strongly inhibited by 1 mM Nethylmaleimide and iodoacetic acid, by 10 mM Hg2+ and Ag+, and was moderately stimulated by Ca2+ and Mg2+.

Ion Decomposition versus Molecular Size Probed by Vacuum Ultraviolet Photoionization

The molecular size dependence of primary fragmentation is studied for a series of β-naphthyl esters having alkyl chain lengths from C 2 to C 18. The esters are vaporized at a known temperature and ionized by coherent vacuum ultraviolet radiation at 10.5 eV. The photoionization wavelength is energetic enough to cause both metastable and nonmetastable primary fragmentation to m/ z 144, but not energetic enough to cause secondary fragmentation to m/ z 115 or 116. Under these conditions, the ratio of the nonmetastable-to-metastable daughter ion current, D/ m D , is expected to give a rough indication of the average parent ion dissociation rate. The D/ m D ratio decreases with increasing molecular size, but not as quickly as expected by simple RRK theory. This behavior along with temperature dependence studies suggests that the internal energy required for dissociation is provided in substantial part by both the initial thermal internal energy and the energy imparted by the photoionization step. The role of thermal energy in the dissociation of large ions is discussed.

Esterases of Lactobacillus helveticus and Lactobacillus delbrueckii ssp. bulgaricus

Intracellular esterase activity of Lactobacillus helveticus CNRZ 32, L. helveticus ATCC 10797, and Lactobacillus delbrueckii ssp. bulgaricus ATCC 12278 were determined using chromogenic o- and p-nitrophenyl derivatives of fatty acids as substrates. Each of the three strains of lactobacilli had active esterase, which hydrolyzed p-nitrophenyl derivatives of acetate, butyrate, and caproate. Lactobacillus delbrueckii ssp. bulgaricus ATCC 12278 had the most esterase activity, followed by L. helveticus CNRZ 32, and then by L. helveticus ATCC 10797. Active esterases in crude cell-free extracts were separated by PAGE; identified by histochemical staining; and their specificities toward α- and β-naphthyl esters of acetic, butyric, caproic, and palmitic acids were determined. Extracts of each of the three lactobacilli exhibited two active esterase bands, but relative mobility values of the enzyme differed among the strains. Esterase activity of L. helveticus CNRZ 32 increased gradually from the beginning of the log phase until it reached a maximum after 24h of incubation. The freezing process reduced, when compared with unfrozen cells, the intracellular esterase activity of L. helveticus CNRZ 32 grown in MRS broth or skim milk. Cells of L. helveticus CNRZ 32 grown in 10% sterile skim milk had slightly less esterase activity (up to 13%), and cells grown in a 7.5% whey-based medium had between 1.3- and 1.7-fold more activity, depending on which test substrate was hydrolyzed, than did MRS-grown cells.

Esterases of Micrococcus Species: Identification and Partial Characterization

Five different strains or species of micrococci were examined for intracellular esterase activity using various chromogenic ortho (o)- and para (p)-nitrophenyl derivatives of fatty acids as substrates. All strains possessed esterase activity, and p-nitrophenyl derivatives of fatty acids were hydrolyzed faster than o-nitrophenyl derivatives by all strains except Micrococcus caseolyticus ATCC 13548, which hydrolyzed o-nitrophenyl propionate faster than p-nitrophenyl propionate and both o- and p-nitrophenyl caprate at an equal rate. Active esterases in cell-free extracts were separated, identified, and their specificity toward various α- and β-naphthyl esters of acetic, propionic, and butyric acids were determined by histochemical staining followed by PAGE. Preparations from all strains exhibited several active bands of esterases on gels, but there was no similarity in relative mobility values of esterases among the strains. The esterase in a crude cell-free extract of Micrococcus species ATCC 8459 was partially characterized using p-nitrophenyl acetate and butyrate as substrates. The esterase had optima at pH 8.0 and 40°C. Esterase activity was inhibited by several divalent metal ions; was strongly inhibited by eserine, diisopropyl fluorophosphate (DFP), and sodium fluoride; and was slightly inhibited by 1,10-phenanthroline and sodium taurocholate. Enzyme activity was markedly inhibited by sodium chloride at pH 7.0 and was completely inhibited at pH 5.0. The activity also was strongly inhibited by reducing and oxidizing agents.

Esterolytic Activities of Pediococcus Species

Six different strains of Pediococcus pentosaceus and two of Pediococcus acidilactici were examined for intracellular esterolytic enzyme activity. All strains of Pediococcus pentosaceus possessed esterase activity, which was highest in Pediococcus pentosaceus NCDO 559 with α-naphthyl acetate as substrate. However, no esterolytic activity was found in either strain of Pediococcus acidilactici tested. Esterolytic enzymes in crude cell-free extracts were separated using PAGE and identified by histochemical staining. Substrate specificity toward various α- and β-naphthyl esters of acetic, propionic, and butyric acids was determined. All strains of Pediococcus pentosaceus exhibited active band(s) of esterase on gels and were most active toward α-naphthyl acetate. No active esterase band was detected in either strain of Pediococcus acidilactici.

Substrate-preference polymorphism at an esterase locus of Drosophila mojavensis.

In a larval esterase of Drosophila mojavensis there are alleles whose products preferentially hydrolyze alpha-naphthyl esters, whereas the majority of the alleles hydrolyze preferentially beta-naphthyl esters. In a collection of laboratory stocks alpha alleles have a frequency of 15%. Three different mobilities of alpha alleles were discovered, suggesting a polymorphism rather than a single mutation event. If substrate-preference polymorphisms are common among "multiple-substrate" enzymes (category II of Gillespie and Langley 1974), allozyme variation at these enzyme loci may well be maintained by balancing selection.

ESTERASES OF BAKER'S YEAST. II. SUBSTRATE SPECIFICITIES TOWARDS ESTERS FORMED DURING SUGAR FERMENTATIONS

Ethyl esters of fatty acids (C2–C12), isoamyl acetate and 2-phenethyl acetate were studied as substrates for yeast esterases and compared with the synthetic substrates, p-nitrophenyl esters and β-naphthyl esters. Intact yeast, the 55% and 55–75% ammonium sulphate precipitate of centrifuged yeast homogenate, and partly purified esterases were used for the determination of the hydrolysation activity towards the esters. The results showed that the yeast esterases prefer to hydrolyse the ethyl esters with acyl chain length of C5 to C12. The acetate esters, ethyl acetate, isoamyl acetate and 2-phenethyl acetate are only very slowly hydrolysed or remain unaffected. The substrate specificity of different esterases varies and can be used for their characterisation. Investigating pH optimum curves using intact yeast and a crude esterase preparation and different substrates confirmed the earlier result that there are esterases on both sides of the plasma membrane. The specificity of intracellular and periplasmically located esterases is, however, different.

A rapid spectrophotometric method for the determination of esterase activity

We have developed a spectrophotometric assay method which continuously records esterase activity at 510 nm by monitoring absorbance changes due to the formation of a diazo dye complex. In our method, α-naphthyl ester substrates are hydrolyzed by enzymatic action to α-naphthol which couples to Fast Blue RR salt (a diazonium salt) forming a diazo dye complex. Our method is unique in directly monitoring the formation of the diazo dye complex without extracting the color of the complex as in other methods that use naphthyl esters and diazo coupling of reaction products. The method appears to be limited to α-naphthyl ester substrates, however, since β-naphthyl esters did not give a linear change in absorbance in the enzymatic reactions tested. With this assay method, one can use a single substrate both to determine esterase units quantitatively in solution and to detect esterase staining activity on gel electrophoresis.

Distribution of Autofluorescence and Esterase and Peroxidase Activities in the Epidermis of Wheat Roots

In the wheat root, peroxidases and esterases specific for a-naphthyl esters of acetate, propionate and butyrate are concentrated in cell walls, particularly the outer wall of epidermal cells undergoing extension. In contrast esterases specific for β-naphthyl esters of propionate and butyrate were intra- cellular and concentrated in epidermal and outer root-cap cells of the wheat root. Both α-naphthyl and β-naphthyl esters of longer-chain fatty acids proved to be poor substrates. The esterases and peroxidases associated with the outer epidermal wall may well be involved in turnover of phenolic acids cross-linked to polysaccharides. In this regard, ferulic acid and diferulate were shown to be constituents of wheat-root cell walls. The distribution of these substances can also be inferred from autofluorescence. Treatment with a commercial pig-liver esterase was without effect on the auto- fluorescence of the root cell-walls. Culture filtrates from Gaeumannomyces graminis did remove significant amounts of autofluorescent wall material. These preparations contained α-naphthyl acetate esterase as well as many polysaccharide hydrolase activities.

Molecular Interaction between E-Prostaglandins and Selected Polymers and Its Potential Utilization in Oral Dosage Form Design

Coacervate formation was observed between some E-prostaglandins and povidone in acetonitrile. This molecular interaction was studied using differential scanning calorimetry, IR spectrophotometry, and light microscopy. The structural requirements for coacervate formation between E-prostaglandins and povidone were investigated. Possible utilization of this molecular interaction in the development of E-prostaglandin formulations was explored. The dissolution rate of some insoluble E-prostaglandin esters increased when they were coprecipitated with povidone and polyethylene glycol. For example, the p-hydroxy-benzaldehyde semicarbazone ester of 16,16-dimethyldinoprostone dissolved about 200 times faster as a povidone coprecipitate than did the control mixture. Enhancement of the dissolution rate was observed for the povidone coprecipitates of dinoprostone and its p-acetylphenyl and β-naphthyl esters but not for the p-phenylphenyl ester. Fast dissolving dispersions of the E-prostaglandin esters also could be prepared with the water-insoluble cross-linked polyvinylpyrrolidone. This type of dispersion was nonglassy and easily dispersible in water. Thus, it might have certain advantages over the classical soluble povidone coprecipitates in terms of ease of handling. The degree of enhancement in dissolution of dispersions of cross-linked polyvinylpyrrolidone and E-prostaglandin esters is apparently dependent on the structure of the esters. The potential dissolution enhancement may be related to the strength of the interaction between the macromolecule and the esters, as indicated by the qualitative relationship between the extent of adsorption of the prostaglandins to cross-linked polyvinylpyrrolidone and the dissolution rate enhancement.