Enhancing the Enantioselectivity and Catalytic Efficiency of Esterase from Bacillus subtilis for Kinetic Resolution of l-Menthol through Semirational Design.

Enzymatic kinetic resolution is a promising way to produce l-menthol. However, the properties of the reported biocatalysts are still unsatisfactory and far from being ready for industrial application. Herein, a para-nitrobenzylesterase (pnbA) gene from Bacillus subtilis was cloned and expressed to produce l-menthol from d,l-menthyl acetate. The highest enantiomeric excess (ee) value of the product generated by pnbA was only approximately 80%, with a high conversion rate (47.8%) of d,l-menthyl acetate with the help of a cosolvent, indicating high catalytic activity but low enantioselectivity (E = 19.95). To enhance the enantioselectivity and catalytic efficiency of pnbA to d,l-menthyl acetate in an organic solvent-free system, site-directed mutagenesis was performed based on the results of molecular docking. The F314E/F315T mutant showed the best catalytic properties (E = 36.25) for d,l-menthyl acetate, with 92.11% ee and 30.58% conversion of d,l-menthyl acetate. To further improve the properties of pnbA, additional mutants were constructed based on the structure-guided triple-code saturation mutagenesis strategy. Finally, four mutants were screened for the best enantioselectivity (ee > 99%, E > 300) and catalytic efficiency at a high substrate concentration (200 g/L) without a cosolvent. This work provides several generally applicable biocatalysts for the industrial production of l-menthol.

Bacteriocin production and technological properties of Enterococcus mundtii and Enterococcus faecium strains isolated from sheep and goat colostrum.

Enterococci are lactic acid bacteria (LAB) that play a role in the aroma formation, maturation, and sensory development of fermented foods such as meat and dairy products. They also contribute to the improvement of the extended shelf life of fermented foods by producing bacteriocin. The aim of this study was to isolate bacteriocin-producing LAB from sheep and goat colostrum, to characterize the bacteriocin-producing strains, and determine the technological properties of the strains. A total of 13 bacteriocin-producing LAB was isolated and identified as 11 Enterococcus mundtii and two Enterococcus faecium. The strains were found to be genetically different from each other by phylogenetic analysis of 16S rRNA gene sequences and random amplified polymorphic-DNA (RAPD-PCR). It has been determined that bacteriocins show activity in a wide pH range and are resistant to heat, lose their activity with proteolytic enzymes and α-amylase, but are resistant to detergents. While the presence of the munKS gene was detected in all of the strains, it was determined that E. faecium HC121.4, HC161.1, E. mundtii HC147.1, HC166.5, and HC166.8 strains contained multiple enterocin genes. Trisin-SDS-PAGE analysis revealed two active protein bands of approximately 5.1 and 5.5 kDa in E. faecium HC121.4 and one active protein band with a weight of approximately 4.96 kDa in other strains. E. mundtii strains and E. faecium HC161.1 were identified as mundticin KS producers, and E. faecium HC121.4 was defined as an enterocin A and B producer. Except for E. mundtii HC166.8, acid production of strains was found to be slow at 6 h and moderate at 24 h. None of them showed extracellular proteolytic and lipolytic activities. It was found that the strains had esterase, esterase lipase, leucine arylamidase, acid phosphatase, and naphthol-AS-Bl-phosphohydrolase activities, while protease activities were low and peptidase activities were high. In conclusion, bacteriocin producer 13 Enterococcus strains isolated from sheep and goat colostrum were found to have the potential to be included in starter culture combinations.

Characterization of the pectin methyl-esterase gene family and its function in controlling pollen tube growth in pear (Pyrus bretschneideri).

Pectin methyl-esterases (PMEs) play crucial roles in plant growth. In this study, we identified 81 PbrPMEs in pear. Whole-genome duplication and purifying selection drove the evolution of PbrPME gene family. The expression of 47 PbrPMEs was detected in pear pollen tube, which were assigned to 13 clusters by an expression tendency analysis. One of the 13 clusters presented opposite expression trends towards the changes of methyl-esterified pectins at the apical cell wall. PbrPMEs were localized in the cytoplasm and plasma membrane. Repression of PbrPME11, PbrPME44, and PbrPME59 resulted in the inhibition of pear pollen tube growth and abnormal deposition of methyl-esterified pectins at pollen tube tip. Pharmacological analysis confirmed that reduced PbrPME activities repressed the pollen tube growth. Overall, we have explored the evolutionary characteristics of PbrPME gene family and found the key PbrPME genes that control the growth of pollen tube, which deepened the understanding of pear fertility regulation.

Genome sequencing of Burkholderia contaminans LTEB11 reveals a lipolytic arsenal of biotechnological interest.

Burkholderia contaminans LTEB11 is a Gram-negative betaproteobacterium isolated as a contaminant of a culture in mineral medium supplemented with vegetable oil. Here, we report the genome sequence of B. contaminans LTEB11, identifying and analyzing the genes involved in its lipolytic machinery and in the production of other biotechnological products.

Profiling of Intra- and Extracellular Enzymes Involved in Fructification of the Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes).

Fruiting bodies of Ganoderma lucidum have been widely used as a source of potent nutraceutical products. However, the key proteins involved in fructifying G. lucidum, to our knowledge, have not yet been reported. We evaluated the protein profile of fruiting and nonfruiting G. lucidum strains at various developmental stages: mycelia, spawn running, pinning, and fruiting body. Four strains of G. lucidum (GL-I to GL-IV) were grown in both liquid medium (mushroom minimal medium broth) and bags of wheat straw, after which the biomass and fruiting bodies were harvested. Enzyme studies revealed enhanced intracellular and extracellular enzymatic activities during the spawn run stage compared with that during mycelial growth in broth. The esterase and peroxidase activities increased significantly during the pinning of the fruiting cultures, thus indicating their positive role in fructification. Fourier transform infrared spectroscopy of proteins at 3 stages of cultivation-spawn run, pin head formation, and fruiting-exhibited the presence of hydrophobic amino acids and an ordered protein structure in fruiting strains (GL-I and GL-II), indicating the presence of hydrophobin proteins and their role in mushroom fructification. However, basic and aromatic amino acids predominated in the nonfruiting strain GL-IV, and an unordered protein structure was present, which indicate the positive role of hydrophobic amino acids and hydrophobin proteins in mushroom fructification.

Heterologous expression, purification and characterization of three novel esterases secreted by the lignocellulolytic fungus Penicillium purpurogenum when grown on sugar beet pulp.

The lignocellulolytic fungus, Penicillium purpurogenum, grows on a variety of natural carbon sources, among them sugar beet pulp. Culture supernatants of P. purpurogenum grown on sugar beet pulp were partially purified and the fractions obtained analyzed for esterase activity by zymograms. The bands with activity on methyl umbelliferyl acetate were subjected to mass spectrometry to identify peptides. The peptides obtained were probed against the proteins deduced from the genome sequence of P. purpurogenum. Eight putative esterases thus identified were chosen for future work. Their cDNAs were expressed in Pichia pastoris. The supernatants of the recombinant clones were assayed for esterase activity, and five of the proteins were active against one or more substrates: methyl umbelliferyl acetate, indoxyl acetate, methyl esterified pectin and fluorescein diacetate. Three of those enzymes were purified, further characterized and subjected to a BLAST search. Based on their amino acid sequence and properties, they were identified as follows: RAE1, pectin acetyl esterase (CAZy family CE 12); FAEA, feruloyl esterase (could not be assigned to a CAZy family) and EAN, acetyl esterase (former CAZy family CE 10).

Small-Molecule Probes Reveal Esterases with Persistent Activity in Dormant and Reactivating Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) is the deadliest bacterial pathogen in the world. An estimated one-third of humans harbor Mtb in a dormant state. These asymptomatic, latent infections impede tuberculosis eradication due to the long-term potential for reactivation. Dormant Mtb has reduced enzymatic activity, but hydrolases that remain active facilitate pathogen survival. We targeted Mtb esterases, a diverse set of enzymes in the serine hydrolase family, and studied their activities using both activity-based probes (ABPs) and fluorogenic esterase substrates. These small-molecule probes revealed functional esterases in active, dormant, and reactivating cultures. Using ABPs, we identified five esterases that remained active in dormant Mtb, including LipM (Rv2284), LipN (Rv2970c), CaeA (Rv2224c), Rv0183, and Rv1683. Three of these, CaeA, Rv0183, and Rv1683, were catalytically active in all three culture conditions. Fluorogenic probes additionally revealed LipH (Rv1399c), Culp1 (Rv1984c), and Rv3036c esterase activity in dormant and active cultures. Esterases with persistent activity are potential diagnostic biomarkers or therapeutic targets for Mtb-infected individuals with latent or active tuberculosis.

BnPME is progressively induced after microspore reprogramming to embryogenesis, correlating with pectin de-esterification and cell differentiation in Brassica napus.

BACKGROUND: Pectins are one of the main components of plant cell walls. They are secreted to the wall as highly methylesterified forms that can be de-esterified by pectin methylesterases (PMEs). The degree of methylesterification of pectins changes during development, PMEs are involved in the cell wall remodeling that occurs during diverse plant developmental processes. Nevertheless, the functional meaning of pectin-related wall remodeling in different cell types and processes remains unclear. In vivo, the microspore follows the gametophytic pathway and differentiates to form the pollen grain. In vitro, the microspore can be reprogrammed by stress treatments becoming a totipotent cell that starts to proliferate and follows the embryogenic pathway, a process known as microspore embryogenesis. RESULTS: To investigate if the change of developmental programme of the microspore towards embryogenesis involves changes in pectin esterification levels, which would cause the cell wall remodeling during the process, in the present study, dynamics of PME expression and degrees of pectin esterification have been analysed during microspore embryogenesis and compared with the gametophytic development, in Brassica napus. A multidisciplinary approach has been adopted including BnPME gene expression analysis by quantitative RT-PCR, fluorescence in situ hybridization, immuno-dot-blot and immunofluorescence with JIM5 and JIM7 antibodies to reveal low and highly-methylesterified pectins. The results showed that cell differentiation at advanced developmental stages involved induction of BnPME expression and pectin de-esterification, processes that were also detected in zygotic embryos, providing additional evidence that microspore embryogenesis mimics zygotic embryogenesis. By contrast, early microspore embryogenesis, totipotency and proliferation were associated with low expression of BnPME and high levels of esterified pectins. CONCLUSIONS: The results show that the change of developmental programme of the microspore involves changes in pectin esterification associated with proliferation and differentiation events, which may cause the cell wall remodeling during the process. The findings indicate pectin-related modifications in the cell wall during microspore embryogenesis, providing new insights into the role of pectin esterification and cell wall configuration in microspore totipotency, embryogenesis induction and progression.

GDSL esterase/lipase genes in Brassica rapa L.: genome-wide identification and expression analysis.

GDSL esterase/lipase proteins (GELPs), a very large subfamily of lipolytic enzymes, have been identified in microbes and many plants, but only a few have been characterized with respect to their roles in growth, development, and stress responses. In Brassica crops, as in many other species, genome-wide systematic analysis and functional studies of these genes are still lacking. As a first step to study their function in B. rapa ssp. pekinensis (Chinese cabbage), we comprehensively identified all GELP genes in the genome. We found a total of 121 Brassica rapa GDSL esterase/lipase protein genes (BrGELPs), forming three clades in the phylogenetic analysis (two major and one minor), with an asymmetrical chromosomal distribution. Most BrGELPs possess four strictly conserved residues (Ser-Gly-Asn-His) in four separate conserved regions, along with short conserved and clade-specific blocks, suggesting functional diversification of these proteins. Detailed expression profiling revealed that BrGELPs were expressed in various tissues, including floral organs, implying that BrGELPs play diverse roles in various tissues and during development. Ten percent of BrGELPs were specifically expressed in fertile buds, rather than male-sterile buds, implying their involvement in pollen development. Analyses of EXL6 (extracellular lipase 6) expression and its co-expressed genes in both B. rapa and Arabidopsis, as well as knockdown of this gene in Arabidopsis, revealed that this gene plays an important role in pollen development in both species. The data described in this study will facilitate future investigations of other BrGELP functions.

Detection of potential suberinase-encoding genes in Streptomyces scabiei strains and other actinobacteria.

Streptomyces scabiei causes common scab, an economically important disease of potato tubers. Some authors have previously suggested that S. scabiei penetration into host plant tissue is facilitated by secretion of esterase enzymes degrading suberin, a lipidic biopolymer of the potato periderm. In the present study, S. scabiei EF-35 showed high esterase activity in suberin-containing media. This strain also exhibited esterase activity in the presence of other biopolymers, such as lignin, cutin, or xylan, but at a much lower level. In an attempt to identify the esterases involved in suberin degradation, translated open reading frames of S. scabiei 87-22 were examined for the presence of protein sequences corresponding to extracellular esterases of S. scabiei FL1 and of the fungus Coprinopsis cinerea VTT D-041011, which have previously been shown to be produced in the presence of suberin. Two putative extracellular suberinase genes, estA and sub1, were identified. The presence of these genes in several actinobacteria was investigated by Southern blot hybridization, and both genes were found in most common-scab-inducing strains. Moreover, reverse transcription - polymerase chain reaction performed with S. scabiei EF-35 showed that estA was expressed in the presence of various biopolymers, including suberin, whereas the sub1 gene appeared to be specifically expressed in the presence of suberin and cutin.

Enhanced biosurfactant production through cloning of three genes and role of esterase in biosurfactant release.

BACKGROUND: Biosurfactants have been reported to utilize a number of immiscible substrates and thereby facilitate the biodegradation of panoply of polyaromatic hydrocarbons. Olive oil is one such carbon source which has been explored by many researchers. However, studying the concomitant production of biosurfactant and esterase enzyme in the presence of olive oil in the Bacillus species and its recombinants is a relatively novel approach. RESULTS: Bacillus species isolated from endosulfan sprayed cashew plantation soil was cultivated on a number of hydrophobic substrates. Olive oil was found to be the best inducer of biosurfactant activity. The protein associated with the release of the biosurfactant was found to be an esterase. There was a twofold increase in the biosurfactant and esterase activities after the successful cloning of the biosurfactant genes from Bacillus subtilis SK320 into E.coli. Multiple sequence alignment showed regions of similarity and conserved sequences between biosurfactant and esterase genes, further confirming the symbiotic correlation between the two. Biosurfactants produced by Bacillus subtilis SK320 and recombinant strains BioS a, BioS b, BioS c were found to be effective emulsifiers, reducing the surface tension of water from 72 dynes/cm to as low as 30.7 dynes/cm. CONCLUSION: The attributes of enhanced biosurfactant and esterase production by hyper-producing recombinant strains have many utilities from industrial viewpoint. This study for the first time has shown a possible association between biosurfactant production and esterase activity in any Bacillus species. Biosurfactant-esterase complex has been found to have powerful emulsification properties, which shows promising bioremediation, hydrocarbon biodegradation and pharmaceutical applications.

The possible role of factor C in common scab disease development.

The Gram-positive soil-borne streptomycetes exhibit a complex life cycle that is controlled by extracellular regulatory molecules. One interesting autoregulator is the protein factor C, originally isolated from the culture fluid of S. albidoflavus 45H. Southern hybridizations and database searches revealed that although homologues of factor C are not present in most Streptomyces strains, an exception is the plant pathogenic S. scabies , which causes common scab disease on potato. In S. scabies and related strains pathogenicity involves a large pathogenicity island that carries thaxtomin biosynthetic genes, the nec1 necrogenic factor and other putative virulence genes. Extracellular enzymes, including extracellular esterases, that attack the surface of the tubers and disintegrate the external protective layer are also known to be involved in pathogenicity. In S. albidoflavus 45H, factor C coordinates the expression of many secreted hydrolases. To find out whether esterase is also regulated by factor C, we made a factor C null mutant of strain 45H. The mutant showed a bald phenotype and was impaired in pathogenicity and esterase activity. This is a first indication that extracellular regulatory factors may play a role in the development of potato scab.

Methyl esterase 1 (StMES1) is required for systemic acquired resistance in potato.

Whether salicylic acid (SA) plays a role in systemic acquired resistance (SAR) signaling in potato is currently unclear because potato, unlike tobacco and Arabidopsis, contains highly elevated levels of endogenous SA. Recent studies have indicated that the SA derivative methyl salicylate (MeSA) serves as a long-distance phloem-mobile SAR signal in tobacco and Arabidopsis. Once in the distal, uninfected tissue of these plant species, MeSA must be converted into biologically active SA by the esterase activity of SA-binding protein 2 (SABP2) in tobacco or members of the AtMES family in Arabidopsis. In this study, we have identified the potato ortholog of tobacco SABP2 (StMES1) and shown that the recombinant protein converts MeSA to SA; this MeSA esterase activity is feedback inhibited by SA or its synthetic analog, 2, 2, 2, 2'-tetra-fluoroacetophenone (tetraFA). Potato plants (cv. Désirée) in which StMES1 activity was suppressed, due to either tetraFA treatment or silencing of StMES1 expression, were compromised for arachidonic acid (AA)-induced SAR development against Phytophthora infestans. Presumably due to the inability of these plants to convert MeSA to SA, the SAR-defective phenotype correlated with elevated levels of MeSA and reduced expression of pathogenesis-related (PR) genes in the untreated distal tissue. Together, these results strongly suggest that SAR signaling in potato requires StMES1, its corresponding MeSA esterase activity, and MeSA. Furthermore, the similarities between SAR signaling in potato, tobacco, and Arabidopsis suggest that at least certain SAR signaling components are conserved among plants, regardless of endogenous SA levels.

Ectopic expression of an esterase, which is a candidate for the unidentified plant cutinase, causes cuticular defects in Arabidopsis thaliana.

Cutinase is an esterase that degrades the polyester cutin, a major component of the plant cuticle. Although cutinase activity has been detected in pollen, the genes encoding this enzyme have not been identified. Here, we report the identification and characterization of Arabidopsis CDEF1 (cuticle destructing factor 1), a novel candidate gene encoding cutinase. CDEF1 encodes a member of the GDSL lipase/esterase family of proteins, although fungal and bacterial cutinases belong to the alpha/beta hydrolase superfamily which is different from the GDSL lipase/esterase family. According to the AtGenExpress microarray data, CDEF1 is predominantly expressed in pollen. The ectopic expression of CDEF1 driven by the 35S promoter caused fusion of organs, including leaves, stems and flowers, and increased surface permeability. Ultrastructural analysis revealed that the cuticle of the transgenic plants was often disrupted and became discontinuous. Subcellular analysis with green fluorescent protein (GFP)-tagged CDEF1 showed that the protein is secreted to the extracellular space in leaves. The recombinant CDEF1 protein has esterase activity. These results are consistent with cutinase being secreted from cells and directly degrading the polyester in the cuticle. CDEF1 promoter activity was detected in mature pollen and pollen tubes, suggesting that CDEF1 is involved in the penetration of the stigma by pollen tubes. Additionally, we found CDEF1 expression at the zone of lateral root emergence, which suggests that CDEF1 degrades cell wall components to facilitate the emergence of the lateral roots. Our findings suggest that CDEF1 is a candidate gene for the unidentified plant cutinase.

[Study on isozymes in six species of Curcuma].

OBJECTIVE: To explore genetic relationships of the 39 materials in six species of Curcuma. METHOD: The peroxidase isozyme (POD) and esterase isozyme (EST) were studied using vertical slab polyacrylamide gel electrophoresis (PAGE) technique, and the zymograms were analyzed using the software of NTSYSpc2. 1. RESULT: The interspecific zymogramatic differences were obvious. Each species possessed its own specific zymogram distinguishing form the others. In the analysis of EST isozyme, C. phaeocaulis, C. wenyujin, C. kwangsiensis and C. chuanhuangjiang had their own specific zymogram. In the analysis of POD isozyme, just C. phaeocaulis and C. kwangsiensis had their specific zymogram. CONCLUSION: The genetic relationships are not associated with the geographical distributions and the genetic relationship between C. longa and C. sichuanensis are very close.

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-beta-glucose, to sinapoyl-l-malate in the seedlings.

Isolation and physiological characterization of Bacillus clausii SKAL-16 isolated from wastewater.

An alkaliphilic bacterium, Bacillus clausii SKAL-16, was isolated from soil that had been contaminated with vegetable oil. The optimal pH and general pH range for bacterial growth was 8, and 7 to 10, respectively. The bacterium could grow on tributyrin and glycerol, but could not grow on acetate and butyrate. The SKAL-16 strain excreted butyric acid during growth on tributyrin, and selectively ingested glycerol during growth on a mixture of butyric acid and glycerol. The SKAL-16 generated intracellular lipase, but did not produce esterase and extracellular lipase. The DNA fragment amplified with the chromosomal DNA of SKAL-16 and primers designed on the basis of the esterase-coding gene of Bacillus clausii KSM-K16 was not identical with the esterase-coding gene contained in the GenBank database. Pyruvate dehydrogenase, isocitrate dehydrogenase, and malate dehydrogenase activities were detected in the cellfree extract (crude enzyme).

Analogous pleiotropic effects of insecticide resistance genotypes in peach-potato aphids and houseflies.

We show that single-point mutations conferring target-site resistance (kdr) to pyrethroids and DDT in aphids and houseflies, and gene amplification conferring metabolic resistance (carboxylesterase) to organophosphates and carbamates in aphids, can have deleterious pleiotropic effects on fitness. Behavioural studies on peach-potato aphids showed that a reduced response to alarm pheromone was associated with both gene amplification and the kdr target-site mutation. In this species, gene amplification was also associated with a decreased propensity to move from senescing leaves to fresh leaves at low temperature. Housefly genotypes possessing the identical kdr mutation were also shown to exhibit behavioural differences in comparison with susceptible insects. In this species, resistant individuals showed no positional preference along a temperature gradient while susceptible genotypes exhibited a strong preference for warmer temperatures.

PaeX, a second pectin acetylesterase of Erwinia chrysanthemi 3937.

Erwinia chrysanthemi causes soft-rot diseases of various plants by enzymatic degradation of the pectin in plant cell walls. Pectin is a complex polysaccharide. The main chain is constituted of galacturonate residues, and some of them are modified by methyl and/or acetyl esterification. Esterases are necessary to remove these modifications and, thus, to facilitate the further degradation of the polysaccharidic chain. In addition to PaeY, the first pectin acetylesterase identified in the E. chrysanthemi strain 3937, we showed that this bacterium produces a second pectin acetylesterase encoded by the gene paeX. The paeX open reading frame encodes a 322-residue precursor protein of 34,940 Da, including a 21-amino-acid signal peptide. Analysis of paeX transcription, by using gene fusions, revealed that it is induced by pectic catabolic products and affected by catabolite repression. The expression of paeX is regulated by the repressor KdgR, which controls all the steps of pectin catabolism; by the repressor PecS, which controls most of the pectinase genes; and by catabolite regulatory protein, the global activator of sugar catabolism. The paeX gene is situated in a cluster of genes involved in the catabolism and transport of pectic oligomers. In induced conditions, the two contiguous genes kdgM, encoding an oligogalacturonate-specific porin, and paeX are both transcribed as an operon from a promoter proximal to kdgM, but transcription of paeX can also be uncoupled from that of kdgM in noninduced conditions. PaeX is homologous to the C-terminal domain of the Butyrivibrio fibriosolvens xylanase XynB and to a few bacterial esterases. PaeX contains the typical box (GxSxG) corresponding to the active site of the large family of serine hydrolases. Purified PaeX releases acetate from various synthetic substrates and from sugar beet pectin. The PaeX activity increased after previous depolymerization and demethylation of pectin, indicating that its preferred substrates are nonmethylated oligogalacturonides. PaeX is mostly found in the periplasmic space of E. chrysanthemi. These data suggest that PaeX is mainly involved in the deacetylation of esterified oligogalacturonides that enter the periplasm by the KdgM porin.

Mouse macrophage paraoxonase 2 activity is increased whereas cellular paraoxonase 3 activity is decreased under oxidative stress.

OBJECTIVE: To determine whether paraoxonases (PONs) are expressed in macrophages and to analyze the oxidative stress effect on their expression and activities. METHODS AND RESULTS: We demonstrated the presence (mRNA, protein, activity) of PON2 and PON3 but not PON1 in murine macrophages, whereas in human macrophages, only PON2 was expressed. Under oxidative stress as present in mouse peritoneal macrophages (MPMs) from apoE-deficient (E0) mice as well as in C57BL6 mice, MPMs that were incubated with buthionine sulfoximine, with angiotensin II, with 7-ketocholesterol, or with oxidized phosphatidylcholine, PON2 mRNA levels and lactonase activity toward dihydrocoumarin significantly increased (by 50% to 130%). In contrast, PON3 lactonase activity toward lovastatin was markedly reduced (by 29% to 57%) compared with control cells. The supplementation of E0 mice with dietary antioxidants (vitamin E, pomegranate juice) significantly increased macrophage PON3 activity (by 23% to 40%), suggesting that oxidative stress was the cause for the reduced macrophage PON3 activity. Incubation of purified PON2 or PON3 with E0 mice MPMs resulted in reduced cellular lipid peroxides content by 14% to 19% and inhibition of cell-mediated LDL oxidation by 32% to 39%. CONCLUSIONS: Increased macrophage PON2 expression under oxidative stress could represent a selective cellular response to reduce oxidative burden, which may lead to attenuation of macrophage foam cell formation.