A cold active (2R,3R)-(−)-di-O-benzoyl-tartrate hydrolyzing esterase from Rhodotorula mucilaginosa

In a screening procedure a pink-colored yeast was isolated from enrichment cultures with (2R,3R)-(−)-di-O-benzoyl-tartrate (benzoyl-tartrate) as the sole carbon source. The organism saar1 was identified by morphological, physiological, and 18S ribosomal DNA/internal transcribed spacer analysis as Rhodotorula mucilaginosa, a basidiomycetous yeast. During growth the yeast hydrolyzed the dibenzoyl ester stoichiometrically to the monoester using the separated benzoate as the growth substrate, before the monoester was further cleaved into benzoate and tartrate, which were both metabolized. The corresponding benzoyl esterase was purified from the culture supernatant and characterized as a monomeric glycosylated 86-kDa protein with an optimum pH of 7.5 and an optimum temperature of 45 °C. At 0 °C the esterase still exhibited 20% of the corresponding activity at 30 °C, which correlates it to psychrophilic enzymes. The esterase could hydrolyze short chain p-nitrophenyl-alkyl esters and several benzoyl esters like benzoyl-methyl ester, ethylene-glycol-dibenzoyl ester, phenyl-benzoyl ester, cocaine, and 1,5-anhydro-d-fructose-tribenzoyl ester. However feruloyl-ethyl ester was not hydrolyzed. The activity characteristics let the enzyme appear as a promising tool for synthesis of benzoylated compounds for pharmaceutical, cosmetic, or fine chemical applications, even at low temperatures.     

Structures of Monohydroperoxides Produced from Chlorophyll Sensitized Photooxidation of Methyl Linoleate

The effects on growth and mortality of larvae of pink bollworm (Pectinophora gossypiella, Saunders), bollworm (Heliothis zea, Boddie) and tobacco budworm (Heliothis virescens, F.) of adding selected C₁₀–C₁₂ fatty acid methyl esters to a standard diet were determined. The antibiotic activity of straight chain saturated esters was compared to the activity of esters with an olefinic bond either at C-2 or terminally or with a terminal acetylenic or cyclopropyl group. The ester with the greatest activity was the naturally occurring compound methyl (Z,Z)-deca-2,8-diene-4,6-diynoate (matricaria ester) which was lethal to all pink bollworm larvae at 0.01% in the diet and lethal to all bollworm and tobacco budworm larvae at 0.05%.     

Characterization of Foam- and Emulsion-stabilizing Functions of Enzymatically Modified Proteins with Surfactancy

A papain-catalyzed reaction involving covalent incorporation of l-leucine n-alkyl ester is available for producing an enzymatically modified protein (EMP) with surfactancy [Agric. Biol. Chem., 45, 1621 (1981)]. In the present work we used gelatin as a starting material and incorporated l-leucine n-hexyl ester to produce a whippable EMP and l-leucine n-dodecyl ester to produce an emulsifiable EMP. A foam system formed with the whippable EMP was much stabler than that formed with sodium dodecylsulfate. The emulsifiable EMP also gave a much stabler oil-in-water emulsion than Tween-80 did. The stability of the emulsion formed with EMP was not affected by the presence of NaCl at a very high concentration. The observed foam and emulsion stabilities were well explained by the data for decreased mobility of the involved water protons. These results may indicate that EMP molecules, when arranged at the air/water or oil/water interface, can bind a part of the water to form thick barriers which prevent the air or oil particles from coalescing.     

Rapid ester biosynthesis screening reveals a high activity alcohol‐O‐acyltransferase (AATase) from tomato fruit

Ethyl and acetate esters are naturally produced in various yeasts, plants, and bacteria. The biosynthetic pathways that produce these esters share a common reaction step, the condensation of acetyl/acyl‐CoA with an alcohol by alcohol‐O‐acetyl/acyltransferase (AATase). Recent metabolic engineering efforts exploit AATase activity to produce fatty acid ethyl esters as potential diesel fuel replacements as well as short‐ and medium‐chain volatile esters as fragrance and flavor compounds. These efforts have been limited by the lack of a rapid screen to quantify ester biosynthesis. Enzyme engineering efforts have also been limited by the lack of a high throughput screen for AATase activity. Here, we developed a high throughput assay for AATase activity and used this assay to discover a high activity AATase from tomato fruit, Solanum lycopersicum (Atf‐S.l). Atf1‐S.l exhibited broad specificity towards acyl‐CoAs with chain length from C₄ to C₁₀ and was specific towards 1‐pentanol. The AATase screen also revealed new acyl‐CoA substrate specificities for Atf1, Atf2, Eht1, and Eeb1 from Saccharomyces cerevisiae, and Atf‐C.m from melon fruit, Cucumis melo, thus increasing the pool of characterized AATases that can be used in ester biosynthesis of ester‐based fragrance and flavor compounds as well as fatty acid ethyl ester biofuels.     

Studies on the distribution and regulation of microbial keto ester reductases

In a limited screening 65 microorganisms were tested with regard to their ability to reduce keto acids or esters of different chain length and position of the keto group with NADH or NADPH as coenzymes. Twenty-seven organisms exhibited reductase activity. Among these, Candida parapsilosis and Rhodococcus erythropolis have been chosen for further investigation. The keto ester reductases of both C. parapsilosis and R. erythropolis prefer NADH as coenzyme and show higher activity towards keto esters than keto acids. The keto ester reductase production of C. parapsilosis during growth passed a maximum in the late exponential phase, decreased and reaches a plateau in the stationary phase. In contrast, the specific activity of the keto ester reductase of R. erythropolis did not decrease in the stationary growth phase. The enzyme of C. parapsilosis was inducible by a keto ester when growing on glycerol as the sole carbon source. Furthermore, the enzyme of C. parapsilosis was subject to catabolite repression. When C. parapsilosis and R. erythropolis were cultivated on n-alcane the specific activity of their keto ester reductases was enhanced about seven- and eightfold, respectively, compared to growth on glucose. This leads to the assumption that, while growing on n-alcane, a degradation product is formed in both strains that induces the production of the keto ester reductase. Correspondence to: M.-R. Kula     

The Synthesis of Some 5-Vinyluracil-Nucleoside Analogues

Abstract

The reaction of 5-iodouridine with esters of acrylic acid in the palladium-catalysed Heck reaction was used to generate a series of esters of the acid (E) -5- (2-carboxyvinyl)uridine in poor to moderate yield. An alternative method starts from the acid by protection of the sugar moiety followed by in situ generation of the acid chloride then ester formation followed by simultaneous sugar deprotection. Treatment of the methyl ester with ammonia and methylamine gave the corresponding amides, while treatment with dimethylamine gave 5-(1-dimethylamino-2-carboxy)ethyluridine as the major product.     

Structure-activity Relationships of Fungicidal N-Benzoylanthranilic Esters

The antifungal activity of 37 N-(methoxy-substituted benzoyl)anthranilic esters was tested on the powdery mildew of barley caused by Erysiphe graminis by the pot test. Among the methyl N-(methoxy-substituted benzoyl)anthranilates tested, 3,4-dimethoxybenzoyl derivative exhibited the highest activity. The variation in fungicidal activity of N-(3,4-dimethoxybenzoyl)anthranilic esters was shown to be related with variation in hydrophobicity and the electronic property of the alcohol moiety of the ester. The branching at the α-position of the alcohol moiety of the ester was detrimental to the activity.     

Triterpene hexahydroxydiphenoyl esters and a quinic acid purpurogallin carbonyl ester from the leaves of Castanopsis fissa

Triterpene hexahydroxydiphenoyl (HHDP) esters have only been isolated from Castanopsis species, and the distribution of these esters in nature is of chemotaxonomical interest. In this study, the chemical constituents of the leaves of Castanopsis fissa were examined in detail to identify and isolate potential HHDP esters. Together with 53 known compounds, 3,4-di-O-galloyl-1-O-purpurogallin carbonyl quinic acid (1) and 3,24-(S)-HHDP-2α,3β,23,24-tetrahydroxytaraxastan-28,20β-olide (2) were isolated and their structures were elucidated by spectroscopic and chemical methods. The polyphenols of the leaves were mainly composed of galloyl quinic acids, triterpenes HHDP esters, ellagitannins and flavonol glycosides. In particular, the isolation yields of 1,3,4-trigalloyl quinic acid and compound 2 were 1.53% and 0.27%, respectively, from the fresh leaves. The presence of lipid soluble HHDP esters of oleanane-type triterpenes as one of the major metabolites is an important chemotaxonomical discovery. Lipase inhibition activities and ORAC values of the major constituents were compared. The triterpene HHDP ester showed moderate lipase inhibition activity and myricitrin gave the largest ORAC value.     

Local anesthetic action of carboxylic esters: Evidence for the significance of molecular volume and for the number of sites involved

Summary The effects of the homologous series of carboxylic esters, methyl propionate to methyl decanoate, on the steadystate inactivation of the sodium current in squid axons have been studied. The esters moved the relationship between the inactivation parameter,h , and the membrane potential in the hyperpolarizing direction, thus reducing the number of sodium channels available at the resting potential. The concentration dependence of the shift at the mid-point of the curve ofh against potential has been measured for all esters except decanoate, which was almost inactive. Two aspects of these concentration dependences suggest that molecular volume is an important determinant of the effectiveness of each ester. Firstly, there is a sharp decline in activity above methyl hexanoate. This cut-off in activity resembles that for hydrocarbons where it has been suggested [e.g., Haydon, D.A., Urban, B.W. 1983)J. Physiol. (London) 341:411–427] to a result from a decrease in uptake with increasing molecular volume. (Further data for the hydrocarbonsn-butane ton-heptane are reported here.) Secondly, the smallest compounds, methyl propionate and methyl butyrate, are less effective than would be predicted if equal membrane concentrations of each ester produced the same shift. The aqueous concentration dependences for these esters indicate that below methyl hexanoate, as the series is descended, progressively higher membrane concentrations are required to produce a given shift. This would be expected if the volume of ester in the membrane, rather than the number of molecules, is important.Differences between the effects of the ester series on steady-state inactivation and on the reduction of the peak sodium current suggest that, in the unclamped squid axon, excitability is influenced by at least two distinct mechanisms in which at least two sites of action are involved.     

Synthesis of fructose esters by Pseudomonas sp. lipase in anhydrous pyridine

Fructose esters were synthesized from fructose and vinyl esters by transesterification catalyzed by lipase AK in anhydrous pyridine. The efficacy of ester synthesis was enhanced by increasing the length of carbon chain in the vinyl ester. Fructose monoesters and diesters were synthesized and their relative production ratio depended on the chain length of vinyl esters. Vinyl esters with chain length longer than C10 produced only monoacyl fructose which has an acyl moiety attached to C1 carbon of fructose. The monoacyl fructose composed of fatty acid with C10 or longer chains had a strong emulsifying activity on various hydrocarbons and oils.     

Adverse effects of extracts of Artocarpus altilis Park,and Azadirachta indica (A. Juss) on the reproductive physiology of the adult female tick,Boophilus microplus (Canest.)

Summary

Adverse effects of extracts from the plants Artocarpus altilis and Azadirachta indica on egg laying and hatching in the tick Boophilus microplus were quantified. A 50% inhibition of egg laying was achieved by a dose of 0.54 and 0.46 μg crude ethanol extract per tick, respectively. These doses also caused a 65% and 80% hatching failure, respectively. Extracts, particularly those of A. indica, inhibit protein and lipid sequestration by ovaries and oocytes. GC-MS analyses revealed reductions in the quantities of four methyl esters sequestered from the ovaries into the oocytes oviposited on the 12th day of engorgement by the treated ticks in the order of (A. indica effects are in parentheses): undecanoic acid 10-methyl-,methyl ester 40% (100%); tetradecanoic acid, methyl ester 100% (100%); tetradecanoic acid, 12-methyl-,methyl ester 100% (100%) and pentadecanoic acid, 14-methyl-,methyl ester 30% (75%).     

Solubilization of low-rank coal by Trichoderma atroviride: Evidence for the involvement of hydrolytic and oxidative enzymes by using 14C-labelled lignite

The deuteromycete Trichoderma atroviride is able to solubilize lignite in dependence on a given carbon source for growth. When cultivated on media containing glutamate, this mold excreted a set of different enzymes with hydrolytic activity. Addition of lignite to the growth media induced the synthesis of extracellular lignite-specific esterase activity but no evidence has been provided for its direct involvement in the process of lignite solubilization. Hence, the basic capability of T. atroviride enzymes to degrade a variety of ester and ether bonds at the surface or within the bulky lignite structure was tested using coal following its direct labelling with ¹⁴C-alkyl iodide. The participation of hydrolytic and oxidative enzymes in lignite degradation was assessed by measuring the release of ¹⁴C radioactivity from selectively alkylated carboxylic and phenolic OH groups. T. atroviride cleaved both carboxylic esters using esterases and the phenolic ether bonds by using oxidative enzymes, most likely laccases. Journal of Industrial Microbiology & Biotechnology (2002) 28, 207–212 DOI: 10.1038/sj/jim/7000232 Received 05 July 2001/ Accepted in revised form 08 December 2001     

Second Generation of cycloSal‐Pronucleotides with Esterase‐Cleavable Sites: The ”Lock‐In”‐Concept

A conceptual extension of the cycloSal‐pronucleotide approach is presented. The characteristic feature of the new cycloSal‐derivatives of the anti‐HIV active nucleoside analogue d4T 1 is the incorporation of an enzymatically cleavable carboxylic ester moiety with the intention to trap the triesters inside cells (”lock‐in”‐concept). CycloSal‐triesters bearing different ester groups in the 3‐or 5‐position of the cycloSal‐moiety are described. Surprisingly, only acetyl‐and levulinyl esters are cleaved readily in CEM cell extracts while alkyl esters were found to be stable. Nevertheless, in in‐vitro anti‐HIV assays most of the compounds achieve the thymidine–kinase bypass, thus proving that they act at least as nucleotide delivery systems.     

Kinetic resolution of 2-chloro-3,3,3-trifluoropropanoic acid esters catalyzed by lipase from Candida rugosa

Abstract

By screening around 30 commercially available lipases and esterases, two enzymes, C. rugosa lipase and P. fluorescens esterase, were found to posess catalytic activity and enantioselectivity (E?10) for the hydrolysis of 2-chloro-3,3,3-trifluoropropanoic acid (CTFPA) methyl and ethyl ester. Both enzymes were tentatively assigned to be (S)-selective based on the assumption that they have the same stereopreference as in the hydrolysis of methyl 2-chloropropanoate, which is a non-fluorinated analogue of CTFPA. The enzymes were applied in the kinetic resolution of CTFPA ethyl ester and 95% ee of the remaining ester could be achieved at 60% conversion. The crosslinked enzyme aggregate (CLEA) of C. rugosa lipase was found to catalyze enantioselective transesterification (E?40) of CTFPA methyl ester with ethanol. By conducting the transesterification in a 10-mL packed-bed reactor containing CLEA, it was possible to convert racemic CTFPA methyl ester into the mixture of (S)-methyl and (R)-ethyl esters with 82% and 90% ee, respectively, at 4.0 g/L^-1/h^-1 space-time yield, which decreased to 1.0 g/L^-1/h^-1 after four repetitive batches.     

Intracellular processing of exogenously derived non-lipoprotein [3H)cholesterol in normal and mutant human skin fibroblasts deficient in acid sterol ester hydrolase

By studying the incorporation and esterification of non-lipoprotein, free [³H]cholesterol in normal and acid sterol ester hydrolase-deficient human fibroblasts, it was examined whether the esterification reaction of the lysosomal acid sterol ester hydrolase contributed to the formation of cellular [³H|cholesteryl esters. Both the normal and the acid sterol ester hydrolase-deficient cells incorporated exogenous, vesicle-derived free [³H]cholesterol linearly as a function of time. Also, the rate of [³H]cholesteryl ester formation was almost the same in normal and mutant fibroblasts, indicating that the apparent esterification activity of the acid sterol ester hydrolase in normal fibroblasts did not contribute to the formation of [³H]cholesteryl esters in intact cells. To examine whether the incorporated [³H]cholesterol was transported into the endoplasmic reticulum and esterified by the acyl-CoA: cholesterol acyltransferase, the rate of [³H]cholesteryl ester formation was measured in the presence or absence of the acyl-CoA: cholesterol acyltransferase-inhibitor 58-035 (Sandoz Inc.). Results showed that the formation of [³H]cholesteryl esters was reduced markedly when cells were co-incubated with the acyltransferase inhibitor. Maximal inhibition (i.e., 75%) was obtained at an inhibitor concentration of 1 μg/ml. Since the inhibitor 58-035 is very specific for acyl-CoA: cholesterol acyltransferase, this finding clearly shows that exogenous, exchangeable [³H]cholesterol can reach and mix with the intracellular substrate pool of the enzyme.     

The Structure of M-Gtfi,An Inhibitor of Glucosyltransferase from S. Mutans,And Its Inhibitory Relationship with Other Sulfate Ester-Containing Inhibitors

Abstract

M-GTFI, an inhibitor of glucosyltransferase from S. mutans was produced by Micromonospora narashinoensis strain No. 731. The isolation procedure for M-GTFI was improved and established for spectro-scopic analyses, and some properties of the inhibitor were investigated. The structure of M-GTFI was shown to be trisodium [2-sulphonato-9-undecenyll-oxacyclotriacont-3-en-2-one, 16, 18-his sulp-honate. The chemical structure of M-GTFI was therefore similar to that of izumenolide which is a β-lactamase inhibitor containing sulfate ester groups in its molecule.

The inhibitory characteristics of M-GTFI were parallel to that of other inhibitory compounds containing sulphate esters but the spectrum of activity was wider.     

Effect of esters based on terpenoids and GABA on fluidity of phospholipid membranes

Abstract

The influence of esters based on gamma-aminobutyric acid (GABA) and mono-/bicyclic terpenoids on membrane structure was investigated. The mechanism of action for terpenoid esters on phospholipids of artificial membranes and lipids isolated from the rat stratum corneum was studied by fluorescence and FT-IR spectroscopy. We report here, that inclusion of monocyclic terpenoid esters in phospholipid liposomes leads to growth of excimer to monomer ratio (I_E/I_M) indicating a decrease of membrane microviscosity. Another mechanism of influence on biomembranes was proposed for ester of bicyclic borneol - in this case a high ratio of vibronic peak intensities (I₁/I₃) was revealed. The addition of terpenoid esters appears in the FT-IR spectra as intensity reduction of absorption bands associated with C?=?O, P?=?O and P–O–С groups of lecithin phospholipids. Similar results were obtained after esters addition to lipids isolated from stratum corneum indicating a decrease of hydrogen bonds number between polar groups of lipids. Thus, the influence of terpenoid esters on molecular organization of the lipid matrix substantiates the feasibility of their use after transdermal delivery in vivo.     

Screening and its potential application of lipolytic activity from a marine environment: characterization of a novel esterase from <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> CL180

To develop an enantioselective lipase/esterase hydrolyzing racemic ofloxacin ester to levofloxacin, samples were collected from a variety of marine environments such as cold sea, hydrothermal vent area, sediment, tidal flat area, arctic sea, marine organisms, and so on. Microorganisms were isolated by plating on an enrichment medium with simultaneous detection of lipolytic activities and screened for the hydrolysis of ofloxacin ester. Three candidates among isolates were selected, and one of them, identified as Yarrowia lipolytica CL180, hydrolyzed preferentially S-enantiomer of racemic ofloxacin ester. The lipase/esterase gene (yli180) was cloned by screening a genomic library. The sequence analysis revealed an open reading frame consisting of 1,431 bp that encoded a protein of 476 amino acids with a molecular mass of 53 kDa. The yli180 gene was expressed in Escherichia coli and purified to homogeneity. The optimum activity of the recombinant protein (rYli180) occurred at pH 7.5 and 35°C, respectively. rYli180 preferentially hydrolyzed p-nitrophenyl esters of fatty acids with short chain lengths of ≤10 carbon atoms. This study represents a novel esterase of type B1 carboxylesterase/lipase family from a marine isolate, showing a potential usage as a biocatalyst because of enantioselectivity toward racemic ofloxacin ester.     

The bioemulsifier alasan: role of protein in maintaining structure and activity

Alasan, the bioemulsifier of Acinetobacter radioresistens KA53, is a high-molecular-mass complex of polysaccharide and protein. Enrichment culture was used to isolate a bacterial strain that grew on alasan as the sole source of carbon and energy, causing the loss of the protein portion of alasan, as well as the emulsifying activity. The degradation was mediated by extracellular proteinases/alasanases. One of these enzymes, referred to as alasanase II, was purified to homogeneity. Alasanase II, as well as pronase, inactivated alasan, whereas a polysaccharide-degrading enzyme mixture, snail juice, had no effect on emulsifying activity. Deproteinization of alasan with phenol yielded a viscous polysaccharide with no emulsifying activity. Heating alasan to 50 °C led to a 2.5-fold irreversible increase in viscosity with no change in emulsifying activity. Heating to 60°–90 °C caused a drop in viscosity and a 5.8-fold increase in emulsifying activity. The deproteinized alasan showed no increase in emulsifying activity and only small changes in viscosity when heated. Received: 31 October 1997 / Accepted: 29 November 1997     

Synthesis of wax esters by a cell-free system from barley (Hordeum vulgare L.)

Experimental evidence for a membranebound microsomal ester synthetase from Bonus barley primary leaves is reported. The results are consistent with at least two mechanisms for the synthesis of barley wax esters: an acyl-CoA-fattyalcohol-transacylase-type reaction and an apparent direct esterification of alcohols with fatty acids. Biosynthesis of wax esters was not specific with regard to the chain length of the tested alcohols. The microsomal preparation readily catalyzed the esterification of C₁₆-, C₁₈-, C₂₂- or C₂₄-labelled alcohols with fatty acids of endogenous origin. Exogenous long-chain alcohols were exclusively incorporated into the alkyl moieties of the esters. Addition of ATP, CoA and-or free fatty acids was not effective in stimulating or depressing the esterifying activity of the microsomal fraction. Partial solubilization of the ester synthetase was obtained using phosphate-buffered saline.Abbreviations P pellet - PBS phosphate-buffered saline - S supernatant - SDS sodium dodecyl sulphate