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Showing papers on "Esterase published in 2015"


Journal ArticleDOI
TL;DR: Esterases from marine metagenomes are cold-adapted enzymes exhibiting broad biochemical diversity reflecting the environmental conditions where they evolved, and can be used for polyester depolymerization.
Abstract: Most of the Earth’s biosphere is cold and is populated by cold-adapted microorganisms. To explore the natural enzyme diversity of these environments and identify new carboxylesterases, we have screened three marine metagenome gene libraries for esterase activity. The screens identified 23 unique active clones, from which five highly active esterases were selected for biochemical characterization. The purified metagenomic esterases exhibited high activity against α-naphthyl and p-nitrophenyl esters with different chain lengths. All five esterases retained high activity at 5 °C indicating that they are cold-adapted enzymes. The activity of MGS0010 increased more than two times in the presence of up to 3.5 M NaCl or KCl, whereas the other four metagenomic esterases were inhibited to various degrees by these salts. The purified enzymes showed different sensitivities to inhibition by solvents and detergents, and the activities of MGS0010, MGS0105 and MGS0109 were stimulated three to five times by the addition of glycerol. Screening of purified esterases against 89 monoester substrates revealed broad substrate profiles with a preference for different esters. The metagenomic esterases also hydrolyzed several polyester substrates including polylactic acid suggesting that they can be used for polyester depolymerization. Thus, esterases from marine metagenomes are cold-adapted enzymes exhibiting broad biochemical diversity reflecting the environmental conditions where they evolved.

75 citations


Journal ArticleDOI
TL;DR: The suggestion that Est_1092 is the enzyme responsible for the degradation of hydroxycinnamic esters on the L. plantarum strains showed great flexibility in their ability to adapt to different environments and growth substrates supports the suggestion that it provides them with additional advantages to survive and grow on plant environments.
Abstract: Lactobacillus plantarum is the lactic acid bacterial species most frequently found in the fermentation of food products of plant origin on which phenolic compounds are abundant. L. plantarum strains showed great flexibility in their ability to adapt to different environments and growth substrates. Of 28 L. plantarum strains analyzed, only cultures from 7 strains were able to hydrolyze hydroxycinnamic esters, such as methyl ferulate or methyl caffeate. As revealed by PCR, only these seven strains possessed the est_1092 gene. When the est_1092 gene was introduced into L. plantarum WCFS1 or L. lactis MG1363, their cultures acquired the ability to degrade hydroxycinnamic esters. These results support the suggestion that Est_1092 is the enzyme responsible for the degradation of hydroxycinnamic esters on the L. plantarum strains analyzed. The Est_1092 protein was recombinantly produced and biochemically characterized. Surprisingly, Est_1092 was able to hydrolyze not only hydroxycinnamic esters, since all the phenolic esters assayed were hydrolyzed. Quantitative PCR experiments revealed that the expression of est_1092 was induced in the presence of methyl ferulate, an hydroxycinnamic ester, but was inhibited on methyl gallate, an hydroxybenzoic ester. As Est_1092 is an enzyme active on a broad range of phenolic esters, simultaneously possessing feruloyl esterase and tannase activities, its presence on some L. plantarum strains provides them with additional advantages to survive and grow on plant environments.

67 citations


Journal ArticleDOI
TL;DR: It is shown that Thermus thermophilus HB27 is an excellent screening host and can be used as an alternative provider of truly novel biocatalysts with industrial relevance for functional metagenomic screening for new enzymes.
Abstract: Functional metagenomic screening strategies, which are independent of known sequence information, can lead to the identification of truly novel genes and enzymes. Since E. coli has been used exhaustively for this purpose as a host, it is important to establish alternative expression hosts and to use them for functional metagenomic screening for new enzymes. In this study we show that Thermus thermophilus HB27 is an excellent screening host and can be used as an alternative provider of truly novel biocatalysts. In a previous study we constructed the mutant strain BL03 that was no longer able to grow on defined minimal medium supplemented with tributyrin as the sole carbon source and could be used as a host to screen for metagenomic DNA fragments that could complement growth on tributyrin. Several thousand single fosmid clones from thermophilic metagenomic libraries from heated compost and hot spring water samples were subjected to a comparative screening for esterase activity in both T. thermophilus strain BL03 and E. coli EPI300. We scored a greater number of active clones in the thermophilic bacterium than in the mesophilic E. coli. From all clones functionally screened in E. coli, only two thermostable α/β-fold hydrolase enzymes with high amino acid sequence similarity to already characterized enzymes were identifiable. In contrast, five further fosmids were found that conferred lipolytic activities in T. thermophilus. Four open reading frames (ORFs) were found which did not share significant similarity to known esterase enzymes. Two of the genes were expressed in both hosts and the novel thermophilic esterases, which based on their primary structures could not be assigned to known esterase or lipase families, were purified and preliminarily characterized. Our work underscores the benefit of using additional screening hosts other than E. coli for the identification of novel biocatalysts with industrial relevance.

66 citations


Journal ArticleDOI
06 Mar 2015-PLOS ONE
TL;DR: The findings present a novel DiBP degradation enzyme and indicate that the purified enzyme may be a promising candidate for DiBP detoxification and for environmental protection.
Abstract: The widely used plasticizer phthalate esters (PAEs) have become a public concern because of their effects on environmental contamination and toxicity on mammals. However, the biodegradation of PAEs, especially diisobutyl phthalate (DiBP), remains poorly understood. In particular, genes involved in the hydrolysis of these compounds were not conclusively identified. In this study, the CarEW gene, which encodes an enzyme that is capable of hydrolyzing ρ-nitrophenyl esters of fatty acids, was cloned from a thermophilic bacterium Bacillus sp. K91 and heterologously expressed in Escherichia coli BL21 using the pEASY-E2 expression system. The enzyme showed a monomeric structure with a molecular mass of approximately 53.76 kDa and pI of 4.88. The enzyme exhibited maximal activity at pH 7.5 and 45°C, with ρ-NP butyrate as the best substrate. The enzyme was fairly stable within the pH range from 7.0 to 8.5. High-pressure liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS) were employed to detect the catabolic pathway of DiBP. Two intermediate products were identified, and a potential biodegradation pathway was proposed. Altogether, our findings present a novel DiBP degradation enzyme and indicate that the purified enzyme may be a promising candidate for DiBP detoxification and for environmental protection.

66 citations


Journal ArticleDOI
TL;DR: Significant alternation in wood chemistry of transgenic plants including an increase in lignin content and S/G ratio, and a decrease in carbohydrate content, and the extent of wood cellulose conversion during hydrolysis after acid pretreatment is improved in the transgenic lines possible due to reduced cell wall cross-links between cell wall biopolymers by PcGCE.

62 citations


Journal ArticleDOI
TL;DR: Data indicated that multiple/cross resistances may have developed in the PR strain to both Bt toxins and conventional insecticides, and consistently reduced ALP provided evidence to support an ALP-mediated Bt resistance mechanism.

60 citations


Journal ArticleDOI
TL;DR: Molecular understanding is expanded of the important role of α‐esterases during the development of resistance to organophosphorous insecticides in B. dorsalis and heterologous expression combined with cytotoxicity assay in Sf9 cells demonstrated that BdCarE4 and Bd carE6 can probably detoxify malathion.
Abstract: Esterase has been reported to be involved in malathion resistance in the oriental fruit fly, Bactrocera dorsalis (Hendel) However, the underlying molecular mechanism of the esterase-mediated resistance remains largely unknown in this species Here, with the use of a strain selected for malathion resistance in the laboratory (MR), we found that two overexpressed α-esterase genes, namely BdCarE4 and BdCarE6, predominant in the adult midgut and fat body, function in conferring malathion resistance in B dorsalis Notably, these two genes were found to be mostly close to the esterase E3, which are usually implicated in detoxifying organophosphate insecticides The transcript levels of BdCarE4 and BdCarE6 were investigated and compared between the MR and a susceptible (MS) strain of B dorsalis Both genes were significantly up-regulated in the MR strain, which was consistent with the enhanced esterase activity in the MR strain However, no changes in either the coding sequence or gene copy number were observed between the two strains Subsequently, heterologous expression combined with cytotoxicity assay in Sf9 cells demonstrated that BdCarE4 and BdCarE6 can probably detoxify malathion Furthermore, RNA interference-mediated knockdown of each of these two genes significantly increased malathion susceptibility in the MR strain adults In conclusion, these results expand our molecular understanding of the important role of α-esterases during the development of resistance to organophosphorous insecticides in B dorsalis

59 citations


Journal ArticleDOI
TL;DR: Isolated lipases/esterases exhibited activity against mostly short- to medium-chain substrates across a range of temperatures and pH, and the function of these novel enzymes recovered in ruminal metabolism needs further investigation, alongside their potential industrial uses.
Abstract: Improving the health beneficial fatty acid content of meat and milk is a major challenge requiring an increased understanding of rumen lipid metabolism. In this study, we isolated and characterized rumen bacterial lipases/esterases using functional metagenomics. Metagenomic libraries were constructed from DNA extracted from strained rumen fluid (SRF), solid-attached bacteria (SAB) and liquid-associated rumen bacteria (LAB), ligated into a fosmid vector and subsequently transformed into an Escherichia coli host. Fosmid libraries consisted of 7,744; 8,448; and 7,680 clones with an average insert size of 30 to 35 kbp for SRF, SAB and LAB, respectively. Transformants were screened on spirit blue agar plates containing tributyrin for lipase/esterase activity. Five SAB and four LAB clones exhibited lipolytic activity, and no positive clones were found in the SRF library. Fosmids from positive clones were pyrosequenced and twelve putative lipase/esterase genes and two phospholipase genes retrieved. Although the derived proteins clustered into diverse esterase and lipase families, a degree of novelty was seen, with homology ranging from 40 to 78 % following BlastP searches. Isolated lipases/esterases exhibited activity against mostly short- to medium-chain substrates across a range of temperatures and pH. The function of these novel enzymes recovered in ruminal metabolism needs further investigation, alongside their potential industrial uses.

58 citations


Journal ArticleDOI
TL;DR: The synthesis of relevant GE model substrates presented here may provide a valuable tool for the screening, selection and development of industrially relevant GEs for delignification of biomass.
Abstract: Lignin-carbohydrate complexes (LCCs) are believed to influence the recalcitrance of lignocellulosic plant material preventing optimal utilization of biomass in e.g. forestry, feed and biofuel applications. The recently emerged carbohydrate esterase (CE) 15 family of glucuronoyl esterases (GEs) has been proposed to degrade ester LCC bonds between glucuronic acids in xylans and lignin alcohols thereby potentially improving delignification of lignocellulosic biomass when applied in conjunction with other cellulases, hemicellulases and oxidoreductases. Herein, we report the synthesis of four new GE model substrates comprising α- and ɣ-arylalkyl esters representative of the lignin part of naturally occurring ester LCCs as well as the cloning and purification of a novel GE from Cerrena unicolor (CuGE). Together with a known GE from Schizophyllum commune (ScGE), CuGE was biochemically characterized by means of Michaelis–Menten kinetics with respect to substrate specificity using the synthesized compounds. For both enzymes, a strong preference for 4-O-methyl glucuronoyl esters rather than unsubstituted glucuronoyl esters was observed. Moreover, we found that α-arylalkyl esters of methyl α-D-glucuronic acid are more easily cleaved by GEs than their corresponding ɣ-arylalkyl esters. Furthermore, our results suggest a preference of CuGE for glucuronoyl esters of bulky alcohols supporting the suggested biological action of GEs on LCCs. The synthesis of relevant GE model substrates presented here may provide a valuable tool for the screening, selection and development of industrially relevant GEs for delignification of biomass. Biotechnol. Bioeng. 2015;112: 914–922. © 2014 Wiley Periodicals, Inc.

53 citations


Journal ArticleDOI
TL;DR: A new design for fluorescence probes of esterase activity that features a carboxylate-side pro-fluorophore is demonstrated with boron dipyrromethene (BODIPY)-based probes 1’a and 1 b, which exhibit a stability to background hydrolysis that is far superior to classical alcohol-side profluorophile-based probes.
Abstract: A new design for fluorescence probes of esterase activity that features a carboxylate-side pro-fluorophore is demonstrated with boron dipyrromethene (BODIPY)-based probes 1 a and 1 b. Because the design relies on the enzyme-catalyzed hydrolysis of an ester group that is not electronically activated, these probes exhibit a stability to background hydrolysis that is far superior to classical alcohol-side profluorophore-based probes, large signal-to-noise ratios, reduced sensitivity to pH variations, and high enzymatic reactivity. The utility of probe 1 a was established with a real-time fluorescence imaging experiment of endogenous esterase activity that does not require washing of the extracellular medium.

53 citations


Journal ArticleDOI
TL;DR: In this paper, the chemical hydrolysis products from Impranil using 0.1 M HCl or 0. 1 M NaOH were identified and compared to the concentration of hydrolysis products formed using three commercial enzymes by proton nuclear magnetic spectroscopy (1H NMR) and Fourier transform infrared spectrograph (FT-IR).

Journal ArticleDOI
TL;DR: The enzymes from MRL-TL could degrade various aliphatic polyesters; therefore, it might be applied for bioremediation in the polyesters-contaminated environments.

Journal ArticleDOI
TL;DR: Results suggest that the higher the distribution ratio of native PBSA-degrading fungi in the soil, the faster the film degradation is, due to the rapid accumulation of secreted esterases in these soils.
Abstract: The relationship between degradation speed of soil-buried biodegradable polyester film in a farmland and the characteristics of the predominant polyester-degrading soil microorganisms and enzymes were investigated to determine the BP-degrading ability of cultivated soils through characterization of the basal microbial activities and their transition in soils during BP film degradation. Degradation of poly(butylene succinate-co-adipate) (PBSA) film was evaluated in soil samples from different cultivated fields in Japan for 4 weeks. Both the degradation speed of the PBSA film and the esterase activity were found to be correlated with the ratio of colonies that produced clear zone on fungal minimum medium-agarose plate with emulsified PBSA to the total number colonies counted. Time-dependent change in viable counts of the PBSA-degrading fungi and esterase activities were monitored in soils where buried films showed the most and the least degree of degradation. During the degradation of PBSA film, the viable counts of the PBSA-degrading fungi and the esterase activities in soils, which adhered to the PBSA film, increased with time. The soil, where the film was degraded the fastest, recorded large PBSA-degrading fungal population and showed high esterase activity compared with the other soil samples throughout the incubation period. Meanwhile, esterase activity and viable counts of PBSA-degrading fungi were found to be stable in soils without PBSA film. These results suggest that the higher the distribution ratio of native PBSA-degrading fungi in the soil, the faster the film degradation is. This could be due to the rapid accumulation of secreted esterases in these soils.

Journal ArticleDOI
TL;DR: Results demonstrate that increased esterase hydrolysis activity, combined with elevated cytochrome P450 monooxygenase detoxicatication, plays an important role in the high levels of lambda-cyhalothrin resistance and can cause cross-resistance to other insecticides in the CRR strain.

Journal ArticleDOI
TL;DR: It is suggested that SFAR4 plays an important role in fatty acid degradation, thus reducing the fatty acid content in Arabidopsis.
Abstract: SFARs (seed fatty acid reducers) belonging to the GDSL lipases/esterases family have been reported to reduce fatty acid storage and composition in mature Arabidopsis seeds. GDSL lipases/esterases are hydrolytic enzymes that possess multifunctional properties, such as broad substrate specificity, regiospecificity, and stereoselectivity. Studies on the physiological functions and biochemical characteristics of GDSL lipases/esterases in plants are limited, so it is important to elucidate the molecular functions of GDSL-type genes. We found that SFAR4 (At3g48460), a fatty acid reducer belonging to the Arabidopsis GDSL lipases/esterases family, was intensely expressed in embryo protrusion, early seedlings, and pollen. The characterization of recombinant SFAR4 protein indicated that it has short-length p-nitrophenyl esterase activity. In addition, SFAR4 enhanced the expression of genes involved in fatty acid metabolism during seed germination and seedling development. SFAR4 elevated the expression of COMATOSE, which transports fatty acids into peroxisomes, and of LACS6 and LACS7, which deliver long-chain acetyl-CoA for β-oxidation. Furthermore, SFAR4 increased the transcription of PED1 and PNC1, which function in importing peroxisomal ATP required for fatty acid degradation. SFAR4 has another function on tolerance to high glucose concentrations but had no significant effects on the expression of the glucose sensor HXK1. The results demonstrated that SFAR4 is a GDSL-type esterase involved in fatty acid metabolism during post-germination and seedling development in Arabidopsis. We suggested that SFAR4 plays an important role in fatty acid degradation, thus reducing the fatty acid content.

Journal ArticleDOI
TL;DR: The results obtained indicate that the new fungal protease preparations AFP and FPII, bacterial protease preparation HT and the new source of fungal Protease preparation F60K have potential for use in meat tenderising applications.

Journal ArticleDOI
TL;DR: Three carboxyl esterases from microbial communities inhabiting the R. exoculata gill that were isolated by naive screens of a gill chamber metagenomic library are identified and biochemically characterized.
Abstract: The shrimp Rimicaris exoculata dominates the fauna in deep-sea hydrothermal vent sites along the Mid-Atlantic Ridge (depth, 2,320 m). Here, we identified and biochemically characterized three carboxyl esterases from microbial communities inhabiting the R. exoculata gill that were isolated by naive screens of a gill chamber metagenomic library. These proteins exhibit low to moderate identity to known esterase sequences ( = 52%) and to each other (11.9 to 63.7%) and appear to have originated from unknown species or from genera of Proteobacteria related to Thiothrix/Leucothrix (MGS-RG1/RG2) and to the Rhodobacteraceae group (MGS-RG3). A library of 131 esters and 31 additional esterase/lipase preparations was used to evaluate the activity profiles of these enzymes. All 3 of these enzymes had greater esterase than lipase activity and exhibited specific activities with ester substrates ( = 356 U mg(-1)) in the range of similar enzymes. MGS-RG3 was inhibited by salts and pressure and had a low optimal temperature (30 degrees C), and its substrate profile clustered within a group of low-activity and substrate-restricted marine enzymes. In contrast, MGS-RG1 and MGS-RG2 were most active at 45 to 50 degrees C and were salt activated and barotolerant. They also exhibited wider substrate profiles that were close to those of highly active promiscuous enzymes from a marine hydrothermal vent (MGS-RG2) and from a cold brackish lake (MGS-RG1). The data presented are discussed in the context of promoting the examination of enzyme activities of taxa found in habitats that have been neglected for enzyme prospecting; the enzymes found in these taxa may reflect distinct habitat-specific adaptations and may constitute new sources of rare reaction specificities.

Journal ArticleDOI
TL;DR: Polygalacturonase as mentioned in this paper is a commercially available pectin degrading enzyme preparation that may contain several other auxiliary enzymes including Pectin methyl esterase and pectins lyase with reasonably trace quantities of cellulases as well.

Journal ArticleDOI
TL;DR: A new robust enzyme with remarkable properties like cold-adaptability, exceptional tolerance to salt and organic solvents provides a promising candidate to meet the needs of some harsh industrial processes.

Journal ArticleDOI
TL;DR: Two fluorogenic esterase probes derived from the far‐red fluorophore 7‐hydroxy‐9H‐(1,3‐dichloro‐9,9‐dimethylacridin‐2‐one) (DDAO) are described and used to reveal ester enzyme activity in protein gel‐resolved mycobacterial lysates.
Abstract: Fluorogenic enzyme probes go from a dark to a bright state following hydrolysis and can provide a sensitive, real-time readout of enzyme activity. They are useful for examining enzymatic activity in bacteria, including the human pathogen Mycobacterium tuberculosis. Herein, we describe two fluorogenic esterase probes derived from the far-red fluorophore 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO). These probes offer enhanced optical properties compared to existing esterase probes because the hydrolysis product, DDAO, excites above 600 nm while retaining a good quantum yield (ϕ=0.40). We validated both probes with a panel of commercially available enzymes alongside known resorufin- and fluorescein-derived esterase substrates. Furthermore, we used these probes to reveal esterase activity in protein gel-resolved mycobacterial lysates. These probes represent new tools for esterase detection and characterization and should find use in a variety of applications.

Journal ArticleDOI
TL;DR: It is revealed that esterase and mixed function oxidase may be involved in cypermethrin resistance in CypRes strain, and overexpression of CYP4M44, CYP9A77 and CYP6B47 can confer metabolic resistance in the Cyp Res strain.

Journal ArticleDOI
TL;DR: The determination of carbofuran in spiked water samples using the proposed biosensor was satisfactory when compared to the chromatographic reference method and showed no significant difference at the 95% confidence level with t-test statistics.

Journal ArticleDOI
Jinyeong Kim1, Seung-Bum Kim1, Sangyoung Yoon1, Eunsoo Hong1, Yeon-Woo Ryu1 
TL;DR: It is found that the mutants generated showed similar thermostability and resistance to additives or organic solvents to Est-AF, without a significant trade-off between activity and stability.
Abstract: Thermostable esterases have potential applications in various biotechnology industries because of their resistance to high temperature and organic solvents. In a previous study, we isolated an esterase from Archaeoglobus fulgidus DSM 4304 (Est-AF), which showed high thermostability but low enantioselectivity toward (S)-ketoprofen ethyl ester. (R)-ketoprofenor (S)-ketoprofenis produced by esterase hydrolysis of the ester bond of (R,S)-ketoprofen ethyl ester and (S)-ketoprofen has better pharmaceutical activity and lower side effects than (R)-ketoprofen. Therefore, we have generated mutants of Est-AF that retained high thermostability whilst improving enantioselectivity. A library of Est-AF mutants was created by error-prone polymerase chain reaction, and mutants with improved enantioselectivity were isolated by site-saturation mutagenesis. The regions of Est-AF containing amino acid mutations were analyzed by homology modeling of its three-dimensional structure, and structure-based explanations for the changes in enantioselectivity are proposed. Finally, we isolated two mutants showing improved enantioselectivity over Est-AF (ee% = −16.2 ± 0.2 and E = 0.7 ± 0.0): V138G (ee% = 35.9 ± 1.0 and E = 3.0 ± 0.1) and V138G/L200R (ee% = 89.2 ± 0.2 and E = 19.5 ± 0.5). We also investigated various characteristics of these mutants and found that the mutants showed similar thermostability and resistance to additives or organic solvents to Est-AF, without a significant trade-off between activity and stability.

Journal ArticleDOI
TL;DR: Chronic effects of the ionic liquid [C4mim][Cl] towards the microalga, Scenedesmus quadricauda were studied by flow cytometry, monitoring multiple endpoints of cell density, esterase activity, membrane integrity, reactive oxygen species and chlorophyll fluorescence.

Journal ArticleDOI
TL;DR: Tenoxicam, fluorometholone acetate, and dexamethasone showed potent inhibitory effects on esterase activity of h CA‐I and hCA‐II isozymes under in vitro conditions.
Abstract: Carbonic anhydrases (CAs) are known as a drug-target enzymes. The inhibitors of the enzyme are important compounds for discovering new therapeutic agents and understanding in detail protein-drug interactions at the molecular level. For this purpose, the in vitro effects of some anti-inflammatory agents such as tenoxicam, fluorometholone acetate, and dexamethasone were investigated on esterase activity of human erythrocyte CA-I and CA-II in this study. hCA-I and hCA-II were purified by affinity chromatography with a yield of 47.25% and 87%, and a specific activity of 642.8 EU/mg proteins and 5576.9 EU/mg proteins, respectively. SDS-PAGE was performed to determine the purity of the enzymes. Inhibitory effects of the drugs on hCA-I and hCA-II were determined by spectrophotometric method. IC50 values for hCA-I and hCA-II were 0.198, 2.18, 11.7, 0.11, 17.5 and 14 μm using tenoxicam, fluorometholone acetate, and dexamethasone, respectively. For fluorometholone acetate and dexamethasone, Ki values from Lineweaver-Burk plots were obtained as 1.044 and 21.2 μm (noncompetitive) for hCA-I and 9.98 and 8.66 μm (non-competitive) for hCA-II. In conclusion, tenoxicam, fluorometholone acetate, and dexamethasone showed potent inhibitory effects on esterase activity of hCA-I and hCA-II isozymes under in vitro conditions.

Journal ArticleDOI
TL;DR: Degradation of both polymer types resulted in a 5-10-fold increase of toxicity of culture supernatans measured with Vibrio fischeri bioluminescence test and Sinapis alba germination plant test, as compared to the biotic and abiotic controls.

Journal ArticleDOI
TL;DR: Comparison of TtEst with structurally related enzymes provides insight into how differences in their catalytic activity can be rationalized based upon the properties of the amino acid residues in their active site pockets.
Abstract: Thermogutta terrifontis esterase (TtEst), a carboxyl esterase identified in the novel thermophilic bacterium T. terrifontis from the phylum Planctomycetes, has been cloned and over-expressed in Escherichia coli. The enzyme has been characterized biochemically and shown to have activity towards small p-nitrophenyl (pNP) carboxylic esters, with optimal activity for pNP-propionate. The enzyme retained 95% activity after incubation for 1 h at 80 °C. The crystal structures of the native TtEst and its complexes with the substrate analogue d-malate and the product acetate have been determined to high resolution. The bound ligands have allowed the identification of the carboxyl and alcohol binding pockets in the enzyme active site. Comparison of TtEst with structurally related enzymes provides insight into how differences in their catalytic activity can be rationalized based upon the properties of the amino acid residues in their active site pockets. The mutant enzymes L37A and L251A have been constructed to extend the substrate range of TtEst towards the larger butyrate and valerate pNP-esters. These mutant enzymes have also shown a significant increase in activity towards acetate and propionate pNP esters. A crystal structure of the L37A mutant was determined with the butyrate product bound in the carboxyl pocket of the active site. The mutant structure shows an expansion of the pocket that binds the substrate carboxyl group, which is consistent with the observed increase in activity towards pNP-butyrate. Database The GenBank sequence accession number for the Thermogutta terrifontis esterase is KR002593. The protein structures for T. terrifontis esterase and their complexes have been deposited in the Protein Data Bank with codes: 4UHC (native), 4UHD (acetate bound), 4UHE (malate bound) and 4UHF (L37A mutant with butyrate bound).

Journal ArticleDOI
27 Jul 2015-PLOS ONE
TL;DR: It was demonstrated that a metagenomic approach is suitable for discovering the lipolytic enzyme diversity and that Est16 has the biotechnological potential for use in industrial processes.
Abstract: Lipolytic enzymes have attracted attention from a global market because they show enormous biotechnological potential for applications such as detergent production, leather processing, cosmetics production, and use in perfumes and biodiesel. Due to the intense demand for biocatalysts, a metagenomic approach provides methods of identifying new enzymes. In this study, an esterase designated as Est16 was selected from 4224 clones of a fosmid metagenomic library, revealing an 87% amino acid identity with an esterase/lipase (accession number ADM63076.1) from an uncultured bacterium. Phylogenetic studies showed that the enzyme belongs to family V of bacterial lipolytic enzymes and has sequence and structural similarities with an aryl-esterase from Pseudomonas fluorescens and a patented Anti-Kazlauskas lipase (patent number US20050153404). The protein was expressed and purified as a highly soluble, thermally stable enzyme that showed a preference for basic pH. Est16 exhibited activity toward a wide range of substrates and the highest catalytic efficiency against p-nitrophenyl butyrate and p-nitrophenyl valerate. Est16 also showed tolerance to the presence of organic solvents, detergents and metals. Based on molecular modeling, we showed that the large alpha-beta domain is conserved in the patented enzymes but not the substrate pocket. Here, it was demonstrated that a metagenomic approach is suitable for discovering the lipolytic enzyme diversity and that Est16 has the biotechnological potential for use in industrial processes.

Journal ArticleDOI
27 Jan 2015-Sensors
TL;DR: A lab-made sequential injection analysis system to analyze off-line samples from shake flasks is proposed, promising to monitor lipase/esterase activity in real time, in which optimization and control strategies can be designed.
Abstract: Lipases and esterases are biocatalysts used at the laboratory and industrial level. To obtain the maximum yield in a bioprocess, it is important to measure key variables, such as enzymatic activity. The conventional method for monitoring hydrolytic activity is to take out a sample from the bioreactor to be analyzed off-line at the laboratory. The disadvantage of this approach is the long time required to recover the information from the process, hindering the possibility to develop control systems. New strategies to monitor lipase/esterase activity are necessary. In this context and in the first approach, we proposed a lab-made sequential injection analysis system to analyze off-line samples from shake flasks. Lipase/esterase activity was determined using p-nitrophenyl butyrate as the substrate. The sequential injection analysis allowed us to measure the hydrolytic activity from a sample without dilution in a linear range from 0.05–1.60 U/mL, with the capability to reach sample dilutions up to 1000 times, a sampling frequency of five samples/h, with a kinetic reaction of 5 min and a relative standard deviation of 8.75%. The results are promising to monitor lipase/esterase activity in real time, in which optimization and control strategies can be designed.

Journal ArticleDOI
TL;DR: Two genes encoding lipolytic enzymes were isolated from a metagenomic library constructed from oil-polluted mud flats and the ability of the enzymes to retain activities in the presence of methanol and the substrates may offer a merit to the biotechnological applications of the proteins.
Abstract: Two genes encoding lipolytic enzymes were isolated from a metagenomic library constructed from oil-polluted mud flats. An esterase gene, est3K, encoded a protein of 299 amino acids (ca. 32,364 Da). Est3K was a family IV esterase with typical motifs, HGGG, and HGF. Although est3K showed high identity to many genes with no information on their enzymatic properties, Est3K showed the highest identity (36 %) to SBLip5.1 from forest soil metagenome when compared to the enzymes with reported properties. A lipase gene, lip3K, encoded a protein of 616 amino acids (ca. 64,408 Da). Lip3K belonged to family I.3 lipase with a C-terminal secretion signal and showed the highest identity (93 %) to the lipase of Pseudomonas sp. MIS38. The presence of several newly identified conserved motifs in Est3K and Lip3K are suggested. Both Est3K and Lip3K exerted their maximal activity at pH 9.0 and 50 °C. The activity of Lip3K was significantly increased by the presence of 30 % methanol. The ability of the enzymes to retain activities in the presence of methanol and the substrates may offer a merit to the biotechnological applications of the enzymes such as transesterification. The activity and the thermostability of Lip3K were increased by Ca2+. Est3K and Lip3K preferred p-nitrophenyl butyrate (C4) and octanoate (C8), respectively, as the substrate and acted independently on the substrates with no synergistic effect.