Role of a carboxylesterase in herbicide bioactivation in Arabidopsis thaliana

Arabidopsis thaliana contains multiple carboxyesterases (AtCXEs) with activities toward xenobiotics, including herbicide esters that are activated to their phytotoxic acids upon hydrolysis. On the basis of their susceptibility to inhibition by organophosphates, these AtCXEs are all serine hydrolases. Using a trifunctional probe bearing a fluorophosphonate together with biotin and rhodamine to facilitate detection and recovery, four dominant serine hydrolases were identified in the proteome of Arabidopsis. Using a combination of protein purification, capture with the trifunctional probe and proteomics, one of these hydrolases, AtCXE12, was shown to be the major carboxyesterase responsible for hydrolyzing the pro-herbicide methyl-2,4-dichlorophenoxyacetate (2,4-D-methyl) to the phytotoxic acid 2,4-dichlorophenoxyacetic acid. Recombinant expression of the other identified hydrolases showed that AtCXE12 was unique in hydrolyzing 2,4-D-methyl. To determine the importance of AtCXE12 in herbicide metabolism and efficacy, the respective tDNA knock-out (atcxe12) plants were characterized and shown to lack expression of AtCXE12 and have greatly reduced levels of 2,4-D-methyl-hydrolyzing activity. Young atcxe12 seedlings were less sensitive than wild type plants to 2,4-D-methyl, confirming a role for the enzyme in herbicide bioactivation in Arabidopsis.     

Carboxylesterase activities toward pesticide esters in crops and weeds

Proteins were extracted from maize, rice, sorghum, soybean, flax and lucerne; the weeds Abutilon theophrasti, Echinochloa crus-galli, Phalaris canariensis, Setaria faberii, Setaria viridis, Sorghum halepense and the model plant Arabidopsis thaliana and assayed for carboxylesterase activity toward a range of xenobiotics. These included the pro-herbicidal esters clodinafop-propargyl, fenoxaprop-ethyl, fenthioprop-ethyl, methyl-2,4-dichlorophenoxyacetic acid (2,4-d-methyl), bromoxynil-octanoate, the herbicide-safener cloquintocet-mexyl and the pyrethroid insecticide permethrin. Highest activities were recorded with alpha-naphthyl acetate and methylumbelliferyl acetate. Esters of p-nitrophenol were also readily hydrolysed, with turnover declining as the chain length of the acyl component increased. Activities determined with model substrates were much higher than those observed with pesticide esters and were of limited value in predicting the relative rates of hydrolysis of the crop protection agents. Substrate preferences with the herbicides were typically 2,4-d-methyl>clodinafop-propargyl>fenthioprop-ethyl, fenoxaprop-ethyl and bromoxynil-octanoate. Isoelectric focussing in conjunction with staining for esterase activity using alpha-naphthyl acetate as substrate confirmed the presence of multiple carboxylesterase isoenzymes in each plant, with major qualitative differences observed between species. The presence of serine hydrolases among the resolved isoenzymes was confirmed through their selective inhibition by the organophosphate insecticide paraoxon. Our studies identify potentially exploitable differences between crops and weeds in their ability to bioactivate herbicides by enzymic hydrolysis and also highlight the usefulness of Arabidopsis as a plant model to study xenobiotic biotransformation.     

利用结构多样性的对硝基苯酚羧酸酯和脂肪醇乙酸酯评估7个枯草芽胞杆菌酯水解酶的指纹图谱

基于GenBank公布的枯草芽胞杆菌168基因组序列,克隆表达了30个预测的酯水解酶基因。结果发现:其中7个酶对对硝基苯酚酯表现出明显的酯水解活力。它们在α/β水解酶家族中分属5个不同的亚家族。通过显色底物和pH指示剂进行的高通量筛选,分别绘制了这7个酶的底物指纹谱。考察了酶催化手性酯水解反应的对映选择性,结果表明:对硝基苄基酯酶PnbA和S-脱乙酰化酶Cah对手性醇的乙酸酯具有较广的底物谱,而PnbA和羧酸酯酶Nap分别对DL-薄荷醇乙酸酯和2-氯-1-苯乙醇乙酸酯/2-萘乙醇乙酸酯有极好的对映选择性(E>200)。此外,发现酯酶YitV催化2-氯-1-苯乙醇乙酸酯水解的反应遵循反-Kazlauskas规则。     

Purification and some properties of intracellular esterase from Pseudomonas fluorescens

A novel esterase was found in Pseudomonas fluorescens cells and purified to homogeneity as determined by polyacrylamide gel electrophoresis. The esterase was extracted from the cells by freeze-thawing and hypotonic treatment. Purification was achieved by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-cellulose and benzylamine-agarose and then electrophoresis. The enzyme catalyzed the hydrolysis of methyl esters, such as methyl butyrate, but its hydrolyzing activity decreased with increase in the chain length of the alcohol moiety, and it did not catalyze the hydrolysis of triacylglycerols, such as triacetin. In contrast, the enzyme acted on various acyl residues in a series of methyl esters, such as dimethyl succinate, methyl methacrylate, and dimethyl malate. The optimum pH for activity of this enzyme with methyl butyrate was 7.0-8.5. The enzyme was inhibited by phenylmethylsulfonylfluoride. Its molecular weight was estimated as 48,000 by molecular sieve electrophoresis and gel filtration on Sephadex G-150.     

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.     

Novel thermophilic and thermostable lipolytic enzymes from a Thailand hot spring metagenomic library

Functional screening for lipolytic enzymes from a metagenomic library (origin: Jae Sawn hot spring, Thailand) resulted in isolation of a novel patatin-like phospholipase (PLP) and an esterase (Est1). PLP contained four conserved domains similar to other patatin-like proteins with lipid acyl hydrolase activity. Likewise, sequence alignment analysis revealed that Est1 can be classified as a family V bacterial lipolytic enzyme. Both PLP and Est1 were expressed heterologously as soluble proteins in E. coli and exhibited more than 50% of their maximal activities at alkaline pH, of 7-9 and 8-10, respectively. In addition, both enzymes retained more than 50% of maximal activity in the temperature range of 50-75 degrees C, with optimal activity at 70 degrees C and were stable at 70 degrees C for at least 120 min. Both PLP and Est1 exhibited high V(max) toward p-nitrophenyl butyrate. The enzymes had activity toward both short-chain (C(4) and C(5)) and long chain (C(14) and C(16)) fatty acid esters. The isolated enzymes, are therefore, different from other known patatin-like phospholipases and esterases, which usually show no activity for substrates longer than C(10). We suggest that PLP and EstA enzymes are novel and have a; b potential use in industrial applications.     

Role of a GDSL lipase-like protein as sinapine esterase in Brassicaceae

The seeds of most members of the Brassicaceae accumulate high amounts of sinapine (sinapoylcholine) that is rapidly hydrolyzed during early stages of seed germination. One of three isoforms of sinapine esterase activity (BnSCE3) has been isolated from Brassica napus seedlings and subjected to trypsin digestion and spectrometric sequencing. The peptide sequences were used to isolate BnSCE3 cDNA, which was shown to contain an open reading frame of 1170 bp encoding a protein of 389 amino acids, including a leader peptide of 25 amino acids. Sequence comparison identified the protein as the recently cloned BnLIP2, i.e. a GDSL lipase-like protein, which displays high sequence identity to a large number of corresponding plant proteins, including four related Arabidopsis lipases. The enzymes belong to the SGNH protein family, which use a catalytic triad of Ser-Asp-His, with serine as the nucleophile of the GDSL motif. The corresponding B. napus and Arabidopsis genes were heterologously expressed in Nicotiana benthamiana leaves and proved to confer sinapine esterase activity. In addition to sinapine esterase activity, the native B. napus protein (BnSCE3/BnLIP2) showed broad substrate specificity towards various other choline esters, including phosphatidylcholine. This exceptionally broad substrate specificity, which is common to a large number of other GDSL lipases in plants, hampers their functional analysis. However, the data presented here indicate a role for the GDSL lipase-like BnSCE3/BnLIP2 as a sinapine esterase in members of the Brassicaceae, catalyzing hydrolysis of sinapine during seed germination, leading, via 1- O -sinapoyl-β-glucose, to sinapoyl- l -malate in the seedlings.     

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.     

Sinapoyltransferases in the light of molecular evolution

Acylation is a prevalent chemical modification that to a significant extent accounts for the tremendous diversity of plant metabolites. To catalyze acyl transfer reactions, higher plants have evolved acyltransferases that accept β-acetal esters, typically 1-O-glucose esters, as an alternative to the ubiquitously occurring CoA-thioester-dependent enzymes. Shared homology indicates that the β-acetal ester-dependent acyltransferases are derived from a common hydrolytic ancestor of the Serine CarboxyPeptidase (SCP) type, giving rise to the name Serine CarboxyPeptidase-Like (SCPL) acyltransferases. We have analyzed structure–function relationships, reaction mechanism and sequence evolution of Arabidopsis 1-O-sinapoyl-β-glucose:l-malate sinapoyltransferase (AtSMT) and related enzymes to investigate molecular changes required to impart acyltransferase activity to hydrolytic enzymes. AtSMT has maintained the catalytic triad of the hydrolytic ancestor as well as part of the H-bond network for substrate recognition to bind the acyl acceptor l-malate. A Glu/Asp substitution at the amino acid position preceding the catalytic Ser supports binding of the acyl donor 1-O-sinapoyl-β-glucose and was found highly conserved among SCPL acyltransferases. The AtSMT-catalyzed acyl transfer reaction follows a random sequential bi-bi mechanism that requires both substrates 1-O-sinapoyl-β-glucose and l-malate bound in an enzyme donor–acceptor complex to initiate acyl transfer. Together with the strong fixation of the acyl acceptor l-malate, the acquisition of this reaction mechanism favours transacylation over hydrolysis in AtSMT catalysis. The model structure and enzymatic side activities reveal that the AtSMT-mediated acyl transfer proceeds via a short-lived acyl enzyme complex. With regard to evolution, the SCPL acyltransferase clade most likely represents a recent development. The encoding genes are organized in a tandem-arranged cluster with partly overlapping functions. With other enzymes encoded by the respective gene cluster on Arabidopsis chromosome 2, AtSMT shares the enzymatic side activity to disproportionate 1-O-sinapoyl-β-glucoses to produce 1,2-di-O-sinapoyl-β-glucose. In the absence of the acyl acceptor l-malate, a residual esterase activity became obvious as a remnant of the hydrolytic ancestor. With regard to the evolution of Arabidopsis SCPL acyltransferases, our results suggest early neofunctionalization of the hydrolytic ancestor toward acyltransferase activity and acyl donor specificity for 1-O-sinapoyl-β-glucose followed by subfunctionalization to recognize different acyl acceptors.     

Medium chain acyl-thioester hydrolase activity in goat and rabbit mammary gland fatty acid synthetase complexes

The goat mammary gland fatty acid synthetase hydrolysed both medium (C_8:0, C_10:0) and long (C_16:0, C_18:0) chain length acyl CoA esters, whereas the enzyme from rabbit mammary gland only hydrolysed long chain length acyl CoA esters. The medium chain acyl-thioester hydrolase activity of goat mammary gland fatty acid synthetase was much less sensitive to inhibition by phenylmethanesulfonyl-fluorid than the long chain acylthioester hydrolase activity. These results indicate the presence of either two acyl-thioester hydrolases with different specificity or one acyl-thioester hydrolase containing two different active sites.     

The structural basis for the narrow substrate specificity of an acetyl esterase from Thermotoga maritima

Acetyl esterases from carbohydrate esterase family 7 exhibit unusual substrate specificity. These proteins catalyze the cleavage of disparate acetate esters with high efficiency, but are unreactive to larger acyl groups. The structural basis for this distinct selectivity profile is unknown. Here, we investigate a thermostable acetyl esterase (TM0077) from Thermotoga maritima using evolutionary relationships, structural information, fluorescent kinetic measurements, and site directed mutagenesis. We measured the kinetic and structural determinants for this specificity using a diverse series of small molecule enzyme substrates, including novel fluorogenic esters. These experiments identified two hydrophobic plasticity residues (Pro228, and Ile276) surrounding the nucleophilic serine that impart this specificity of TM0077 for small, straight-chain esters. Substitution of these residues with alanine imparts broader specificity to TM0077 for the hydrolysis of longer and bulkier esters. Our results suggest the specificity of acetyl esterases have been finely tuned by evolution to catalyze the removal of acetate groups from diverse substrates, but can be modified by focused amino acid substitutions to yield enzymes capable of cleaving larger ester functionalities.     

Characterization of the folate salvage enzyme p-aminobenzoylglutamate hydrolase in plants

Folates break down in vivo to give pterin and p-aminobenzoylglutamate (pABAGlu) fragments, the latter usually having a polyglutamyl tail. Pilot studies have shown that plants can hydrolyze pABAGlu and its polyglutamates to p-aminobenzoate, a folate biosynthesis precursor. The enzymatic basis of this hydrolysis was further investigated. pABAGlu hydrolase activity was found in all species and organs tested; activity levels implied that the proteins responsible are very rare. The activity was located in cytosol/vacuole and mitochondrial fractions of pea (Pisum sativum L.) leaves, and column chromatography of the activity from Arabidopsis tissues indicated at least three peaks. A major activity peak from Arabidopsis roots was purified 86-fold by a three-column procedure; activity loss during purification exceeded 95%. Size exclusion chromatography gave a molecular mass of approximately 200 kDa. Partially purified preparations showed a pH optimum near 7.5, a Km value for pABAGlu of 370 microM, and activity against folic acid. Activity was relatively insensitive to thiol and serine reagents, but was strongly inhibited by 8-hydroxyquinoline-5-sulfonic acid and stimulated by Mn2+, pointing to a metalloenzyme. The Arabidopsis genome was searched for proteins similar to Pseudomonas carboxypeptidase G, which contains zinc and is the only enzyme yet confirmed to attack pABAGlu. The sole significant matches were auxin conjugate hydrolase family members and the At4g17830 protein. None was found to have significant pABAGlu hydrolase activity, suggesting that this activity resides in hitherto unrecognized enzymes. The finding that Arabidopsis has folate-hydrolyzing activity points to an enzymatic component of folate degradation in plants.     

Neutral and acid retinyl ester hydrolases associated with rat liver microsomes: relationships to microsomal cholesteryl ester hydrolases

We recently reported the presence of a neutral, bile salt-independent retinyl ester hydrolase (REH) activity in rat liver microsomes and showed that it was distinct from the previously studied bile salt-dependent REH and from nonspecific carboxylesterases (Harrison, E. H., and M. Z. Gad. 1989. J. Biol. Chem. 264: 17142-17147). We have now further characterized the hydrolysis of retinyl esters by liver microsomes and have compared the observed activities with those catalyzing the hydrolysis of cholesteryl esters. Microsomes and microsomal subfractions enriched in plasma membranes and endosomes catalyze the hydrolysis of retinyl esters at both neutral and acid pH. The acid and neutral REH enzyme activities can be distinguished from one another on the basis of selective inhibition by metal ions and by irreversible, active site-directed serine esterase inhibitors. The same preparations also catalyze the hydrolysis of cholesteryl esters at both acid and neutral pH. However, the enzyme(s) responsible for the neutral REH activity can be clearly responsible for the neutral REH activity can be clearly differentiated from the neutral cholesteryl ester hydrolase(s) on the basis of differential stability, sensitivity to proteolysis, and sensitivity to active site-directed reagents. These results suggest that the neutral, bile salt-independent REH is relatively specific for the hydrolysis of retinyl esters and thus may play an important physiological role in hepatic vitamin A metabolism. In contrast to the neutral hydrolases, the activities responsible for hydrolysis of retinyl esters and cholesterol esters at acid pH are similar in their responses to the treatments mentioned above. Thus, a single microsomal acid hydrolase may catalyze the hydrolysis of both types of ester.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences between microsomal and mitochondrial-matrix palmitoyl-coenzyme A hydrolase, and palmitoyl-L-carnitine hydrolase from rat liver.

Palmitoyl-CoA hydrolase (EC 3.1.2.2) and palmitoyl-L-carnitine hydrolase (EC 3.1.1.28) activities from rat liver were investigated. 1. Microsomal and mitochondrial-matrix palmitoyl-CoA hydrolase activities had similar pH and temperature optima, although the activities showed different temperature stability. They were inhibited by Pb2+ and Zn2+. The palmitoyl-CoA hydrolase activities in microsomal fraction and mitochondrial matrix were differently affected by the addition of Mg2+, Ca2+, Co2+, K+ and Na+ to the reaction mixture. ATP, ADP and NAD+ stimulated the microsomal activity and inhibited the mitochondrial-matrix enzyme. The activity of both the microsomal and mitochondrial-matrix hydrolase enzymes was specific for long-chain fatty acyl-CoA esters (C12-C18), with the highest activity for palmitoyl-CoA. The apparent Km for palmitoyl-CoA was 47 microM for the microsomal enzyme and 17 microM for the mitochondrial-matrix enzyme. 2. The palmitoyl-CoA hydrolase and palmitoyl-L-carnitine hydrolase activities of microsomal fraction had similar pH optima and were stimulated by dithiothreitol, but were affected differently by the addition of Pb2+, Mg2+, Ca2+, Mn2+ and cysteine. The two enzymes had different temperature-sensitivities. 3. The data strongly suggest that palmitoyl-CoA hydrolase and palmitoyl-L-carnitine hydrolase are separate microsomal enzymes, and that the hydrolysis of palmitoyl-CoA in the microsomal fraction and mitochondria matrix was catalysed by two different enzymes.     

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.     

Enhanced preference for pi-bond containing substrates is correlated to Pro110 in the substrate-binding tunnel of Escherichia coli thioesterase I/protease I/lysophospholipase L(1)

Escherichia coli thioesterase I/protease I/lysophospholipase L(1) (TAP) possesses multifunctional enzyme with thioesterase, esterase, arylesterase, protease, and lysophospholipase activities. Leu109, located at the substrate-binding tunnel, when substituted with proline (Pro) in TAP, shifted the substrate-preference from medium-to-long acyl chains to shorter acyl chains of triglyceride and p-nitrophenyl ester, and increased the preference for aromatic-amino acid-derived esters. In the three-dimensional TAP structures, the only noticeable alteration of backbone and side chain conformation was located at the downstream Pro110-Ala123 region rather than at Pro109 itself. The residue Pro110, adjacent to Leu109 or Pro109, was found to contribute to the substrate preference of TAP enzymes for esters containing acyl groups with pi bond(s) or aromatic group(s). Some of the interactions between the enzyme protein and the substrate may be contributed by an attractive force between the Pro110 C-H donor and the substrate pi-acceptor.     

A cold-active esterase with a substrate preference for vinyl esters from a psychrotroph, Acinetobacter sp. strain no. 6: gene cloning, purification, and characterization

It has recently been shown that fatty acid vinyl esters serve as effective acylating agents for the synthesis of esters by enzymatic transesterification in high yields. To enhance the usefulness of this system at low temperatures, we have searched for the gene coding for a cold-active lipolytic enzyme with a substrate preference for fatty acid vinyl esters and obtained it from the genomic library of Acinetobacter sp. strain no. 6, a psychrotroph isolated from Siberian soil. The gene (termed aelh, 777 bp) encoded a protein of 258 amino acids, and sequence analysis revealed that the enzyme shows a high sequence similarity to β-ketoadipate enol-lactone hydrolase involved in the β-ketoadipate pathway for the bacterial catabolism of benzoic acid. The aelh gene was expressed in the E. coli C600 cells under the control of lac promoter and the expression product was purified to homogeneity and characterized. It was a monomeric esterase preferentially catalyzing the hydrolysis of enol esters, such as fatty acid vinyl esters with a short-chain acyl group. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride, a specific inhibitor for serine hydrolases. The enzyme could also catalyze transesterification, for example, between vinyl propionate and propanol yielding propyl propionate at 4 °C. These results indicate the usefulness of an esterase (termed AELH) for the enzymatic synthesis of esters by transesterification using vinyl esters as an acyl donor.     

Unique regulation of the active site of the serine esterase S-formylglutathione hydrolase

S-Formylglutathione hydrolases (SFGHs) are highly conserved thioesterases present in prokaryotes and eukaryotes, and form part of the formaldehyde detoxification pathway, as well as functioning as xenobiotic-hydrolysing carboxyesterases. As defined by their sensitivity to covalent modification, SFGHs behave as cysteine hydrolases, being inactivated by thiol alkylating agents, while being insensitive to inhibition by organophosphates such as paraoxon. As such, the enzyme has been classified as an esterase D in animals, plants and microbes. While SFGHs do contain a conserved cysteine residue that has been implicated in catalysis, sequence analysis also reveals the classic catalytic triad of a serine hydrolase. Using a combination of selective protein modification and X-ray crystallography, AtSFGH from Arabidopsis thaliana has been shown to be a serine hydrolase rather than a cysteine hydrolase. Uniquely, the conserved reactive cysteine (Cys59) previously implicated in catalysis lies in close proximity to the serine hydrolase triad, serving a gate-keeping function in comprehensively regulating access to the active site. Thus, any covalent modification of Cys59 inhibited all hydrolase activities of the enzyme. When isolated from Escherichia coli, a major proportion of recombinant AtSFGH was recovered with the Cys59 forming a mixed disulfide with glutathione. Reversible disulfide formation with glutathione could be demonstrated to regulate hydrolase activity in vitro. The importance of Cys59 in regulating AtSFGH in planta was demonstrated in transient expression assays in Arabidopsis protoplasts. As determined by fluorescence microscopy, the Cys59Ser mutant enzyme was shown to rapidly hydrolyse 4-methylumbelliferyl acetate in paraoxon-treated cells, while the native enzyme was found to be inactive. Our results clarify the classification of AtSFGHs as hydrolases and suggest that the regulatory and conserved cysteine provides an unusual redox-sensitive regulation to an enzyme functioning in both primary and xenobiotic metabolism in prokaryotes and eukaryotes.     

Purification and characterization of Lip2 and Lip3 isoenzymes from a Candida rugosa pilot-plant scale fed-batch fermentation

Previous purification of a crude extracellular enzyme preparation from Candida rugosa ATCC 14830 pilot-plant fed-batch fermentations showed the presence of two lipase isoenzymes, Lip2 and Lip3, differing in their molecular masses (58 and 62 kDa, respectively). These enzymes were purified but the lipases were forming active aggregates with a molecular mass higher than 200 kDa. In this work we developed a purification method following three steps: ammonium sulfate precipitation, sodium cholate treatment and ethanol/ether precipitation, and anion exchange chromatography which allowed the sequential disaggregation of the isoenzymes. Pure and monomeric Lip2 and Lip3 were characterized according to pI, glycosylation and activity for p-nitrophenol esters and triacylglycerols of varying acyl chain. Lip3 was the best catalyst for the hydrolysis of the simple esters and triacylglycerols with short and medium acyl chains.