Extracellular Esterase Research Articles

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.

Cloning, Expression, and Characterization of a Recombinant Esterase from Cold-Adapted Pseudomonas mandelii

A gene coding for the extracellular esterase (EstK) was cloned from the psychrotrophic bacterium Pseudomonas mandelii based on its partial amino acid sequence as determined by mass spectrometry. The entire open reading frame consisting of 1,011 bp was expressed in Escherichia coli as a soluble protein and purified by nickel-chelated affinity chromatography and Capto Q column chromatography. Here, we show that the 33-kDa recombinant EstK protein (rEstKsp) had a substrate preference for esters of short-chain fatty acids, especially, p-nitrophenyl acetate. Optimum activity of rEstKsp was at pH 8.5 and 40 °C. The esterase activity remained similar from a range of 4∼20 °C, but the maximum activity varied depending upon pH. With p-nitrophenyl acetate as the substrate, K (M) was 210 μM and k (cat) was 3.4 s(-1). Circular dichroism and fluorescence spectroscopy results revealed that rEstKsp had a predominantly α-helical structure and maintained its folded state at 4∼40 °C. Interestingly, the tertiary structure of rEstKsp was predicted based on the structures of other hyperthermophilic esterases. Our results demonstrated that both native and rEstKsp are active at low temperatures and have a unique substrate preference for p-nitrophenyl acetate.

Extracellular and intracellular esterase processing of SCFA–hexosamine analogs: Implications for metabolic glycoengineering and drug delivery

This report provides a synopsis of the esterase processing of short chain fatty acid (SCFA)-derivatized hexosamine analogs used in metabolic glycoengineering by demonstrating that the extracellular hydrolysis of these compounds is comparatively slow (e.g., with a t1/2 of ∼4h to several days) in normal cell culture as well as in high serum concentrations intended to mimic in vivo conditions. Structure–activity relationship (SAR) analysis of common sugar analogs revealed that O-acetylated and N-azido ManNAc derivatives were more refractory against extracellular inactivation by FBS than their butanoylated counterparts consistent with in silico docking simulations of Ac4ManNAc and Bu4ManNAc to human carboxylesterase 1 (hCE1). By contrast, all analogs tested supported increased intracellular sialic acid production within 2h establishing that esterase processing once the analogs are taken up by cells is not rate limiting.

Characterization of an extracellular thermophilic alkaline esterase produced by Bacillus subtilis DR8806

► An alkaline lipolytic metalloenzyme from Bacillus subtilis DR8806 was characterized. ► The optimum pH and temperature of this enzyme were observed to be 8.0 and 50 °C. ► The enzyme showed a preferential specificity for ester of p-nitrophenyl acetate (C 2 ). ► The enzyme exhibited K m of 4.2 mM and V max of 151 μmol min −1 mg −1 to C 2 ester. ► The hydrolysis of fluorescence esters was increased in the presence of the enzyme. This work is a report of the characterization of an alkaline lipolytic enzyme isolated from Bacillus subtilis DR8806. The extracellular extract was concentrated using ammonium sulfate, and ultrafiltration. The active enzyme was purified by Q-sepharose ion exchange chromatography. The molecular mass of the enzyme was estimated to be 60.25 kDa based on SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). The optimum pH and temperature of this enzyme were observed to be 8.0 and 50 °C, respectively. The enzyme exhibited a half-life of 72 min at its optimum temperature. It was stable in the presence of metal ions (10 mM) such as Ca 2+ , K + and Na + , whereas Cu 2+ , Fe 2+ , Zn 2+ , Mn 2+ , Co 2+ , Mg 2+ and Hg 2+ were found to have inhibitory effects. However, the enzyme activity was not affected significantly by 1% Triton X-100. The study of substrate specificity showed that the purified enzyme has a preferential specificity for small ester of p-nitrophenyl acetate (C 2 ), and it was the most efficiently hydrolyzed substrate as compared to the other esters. The kinetic parameters showed that the enzyme has K m of 4.2 mM and V max of 151 μmol min −1 mg −1 for p-nitrophenyl acetate. The hydrolysis rates of the fluorescence substrates were increased in the presence of the purified enzyme. Regarding the features of the enzyme, it may be utilized as a novel candidate for industrial applications.

Erratum to: Purification and properties of an extracellular esterase from a cold-adapted Pseudomonas mandelii

Erratum to: Purification and properties of an extracellular esterase from a cold-adapted Pseudomonas mandelii

Purification and characterization of an extracellular esterase from a moderately halophilic bacterium, Halobacillus sp. strain LY5

A newly moderate halophilic strain LY5 producing extracellular esterase was isolated and identified. Biochemical and physiological characterization, along with 16S rRNA gene sequence analysis placed the isolate in the genus Halobacillus. Esterase production started from the mid-exponential phase of bacterial growth and reached a maximum level during the mid-stationary phase. The enzyme was highly active against p-nitrophenyl esters with acyl chain lengths from C2 to C10, indicating an esterase activity. Enzyme purification was carried out by combination of 80% ammonium sulphate precipitation, diethylaminoethyl (DEAE)-cellulose ion exchange and Sephacryl S-100 gel filtration chromatography. The molecular weight of the esterase was determined to be 96 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme was highly active over broadranges of temperature (30 to 90°C), pH (6.0 to 12.0) and NaCl concentration (0 to 20%), showing optimal activity at 50°C, pH 10.0 and 10% NaCl. Complete inhibition of the esterase by phenylmethylsulfonyl fluoride (PMSF), phenylarsine oxide (PAO) and diethyl pyrocarbonate (DEPC) indicated that serine, cysteine and histidine residues were essential for its catalytic function. Moreover, it exhibited remarkable stability towards SDS and Triton X-100. Results from the present study indicate that the extracellular esterase from Halobacillus sp. LY5 may have considerable potential for industrial application from the perspectives of its properties. Key words: Moderately halophilic bacterium, esterase, purification and characterization, Halobacillus.

Degradation of Polyurethane by Aspergillus flavus (ITCC 6051) Isolated from Soil

The present study deals with the isolation of fungi from soil with the ability to degrade polyurethane (PU). A pure fungal isolate was analyzed for its ability to utilize PU as a sole carbon source in shaking culture for 30 days. Incubation of PU with Aspergillus flavus resulted in 60.6% reduction in weight of PU. The scanning electron microscopy and Fourier transform infrared spectroscopy (FTIR) results showed certain changes on the surface of PU film and formation of some new intermediate products after polymer breakdown. Thermogravimetric curves showed changes between the thermal behavior of the samples that were inoculated with A. flavus and control. FTIR spectra showed detectable changes in control and incubated samples, suggesting that degradation occurs, with the decreased intensity of band at 1,715 cm(-1), corresponding to ester linkages. We have identified an extracellular esterase activity which might be responsible for the polyurethanolytic activity.

Production and some properties of the thermostable feruloyl esterase and xylanase from Bacillus pumilus

This paper reports the enhanced production of extracellular thermostable feruloyl esterase and xylanase from Bacillus pumilus by optimization of inducing carbon sources. Batch studies were evaluated by varying the ratio of xylan and wheat bran on the medium for enzyme production by the bacterial culture. The feruloyl esterase and xylanase production were highest in culture filtrates from B. pumilus grown in media containing 1% xylan and 3% wheat bran, which were enhanced 2.4-fold and 3.6-fold compare to single xylan medium, respectively. The crude feruloyl esterase and xylanase were thermostable with the maximum activities at 50 and 55°C, and pH 5.8 and 7.4, respectively. Activity at temperatures of 40 to 45°C was high and stable, suggesting that these enzymes have a potential application in obtaining ferulic acid from agro-industrial waste materials in this moderate temperature range. Key words: Bacillus pumilus , feruloyl esterase, xylanase, production, partial characterization.

Purification and properties of an extracellular esterase from a cold-adapted Pseudomonas mandelii

An extracellular esterase, EstK, was purified from the psychrotrophic bacterium Pseudomonas mandelii grown at 25°C. Prior to harvest, cells were treated with 0.2 M MgCl2 to precipitate lipopolysaccharides in the outer membranes, which otherwise form aggregates with the secreted enzymes. EstK was purified to homogeneity using standard procedures. It had substrate specificity towards esters of short-chain fatty acids, particularly, p-nitrophenyl acetate. Optimum activity of EstK was at 40°C; at 4°C the activity was ~50% of its maximum. EstK has a unique substrate preference for p-nitrophenyl acetate and remains active at low temperatures.

In Vitro Evaluation of Phospholipase, Proteinase, and Esterase Activities of Candida parapsilosis and Candida metapsilosis

The aim of this study is to characterize extracellular phospholipase, proteinase, and esterase activities of Candida parapsilosis and C. metapsilosis isolated from clinical sources. Using PCR-restriction fragment length polymorphism (PCR-RFLP) of the secondary alcohol dehydrogenase (SADH) gene fragment, we identified 20 as C. parapsilosis and 11 as C. metapsilosis from 31 isolates of C. parapsilosis species complex. No C. orthopsilosis was identified. A significantly high isolation frequency of C. metapsilosis (35.5%) was observed. Subsequent evaluation of enzymatic profile showed that 90.5% of C. parapsilosis and 91.7% of C. metapsilosis isolates were phospholipase producers. No difference in phospholipase activity was observed between two species. In terms of proteinase, 81.0% of C. parapsilosis and 83.3% of C. metapsilosis isolates were positive. A higher level of proteinase activity was detected in C. parapsilosis. A remarkably high proportion of both C. parapsilosis and C. metapsilosis isolates exhibited strong phospholipase and proteinase activities, suggesting that the production of these two enzymes might be common for them. On the other hand, both species similarly displayed rare esterase activity, with only one C. parapsilosis and two C. metapsilosis isolates being positive. Our data may further add to the confusion concerning the hydrolytic enzymatic activities of the C. parapsilosis complex, and a wider collection of isolates and standardized methods may help to address the issue.

Isolation and characterization of an extracellular thermoalkanophilic P(3HB-co-3HV) depolymerase from Streptomyces sp. IN1

Here, we report on the biodegradation of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] by a novel thermoalkanophilic extracellular esterase from the soil isolate Streptomyces sp. IN1. Preliminary screening and isolation of the bacterium was done using polyhydroxyalkanoate latex medium (PHALM). The isolate was cultured with P(3HB-co-3HV) as the only carbon source and by-products of degradation were derivatized with [N,O-bis(trimethylsilyl)trifluroacetamide] (BSTFA). These products were identified by gas chromatography/mass spectrometry (GC–MS) as silylated hydroxybutyric acid (3HB) and hydroxyvaleric acid, suggesting extracellular depolymerase activity by the isolate. The depolymerase was isolated by (NH 4) 2SO 4 fractionation, dialyzed and purified using fast protein liquid chromatography (FPLC), and confirmed using P(3HB-co-3HV) as a sole source of carbon. The molecular mass of the FPLC purified enzyme occurred between 45 and 66 kDa (SDS–PAGE), but was confirmed by matrix assisted laser desorption ionization–time of flight mass spectrometry (MALDI–TOF MS) to be 62 kDa. Enzyme activity was significantly inhibited by phenylmethylsulfonyl fluoride (PMSF), dithiothreitol (DTT), and Tween 80, but induced by azide (N 3−). Sensitivity to PMSF, DTT, and Tween 80 suggests the involvement of serine as an active site amino acid with disulphide bonds contributing to the catalytic activity, as well as the presence of hydrophobic regions in the enzyme. Non-inhibition of activity by azide indicates that metal ions may not be required as cofactors for activity. This observation was further corroborated by the decrease in enzyme activity in the presence of metal ions such as Ca 2+, Mg 2+, Na +, and K +. The kinetic parameters, V max and K m, in the presence of p-nitrophenylbutyrate as substrate, were determined to be 5.06 × 10 −1 μmol min −1 and 6.73 × 10 −1 mM, respectively.

Production of ferulic acid from lignocellulolytic agricultural biomass by Thermobifida fusca thermostable esterase produced in Yarrowia lipolytica transformant

A gene (axe) encoding the AXE thermostable esterase in Thermobifida fusca NTU22 was cloned into a Yarrowia lipolytica P01g host strain. Recombinant expression resulted in extracellular esterase production at levels as high as 70.94U/ml in Hinton flask culture broth, approximately 140 times higher than observed in a Pichia pastoris expression system. After 72h of fermentation by the Y. lipolytica transformant in the fed-batch fermentor, the fermentation broth accumulated 41.11U/ml esterase activity. Rice bran, wheat bran, bagasse and corncob were used as hydrolysis substrates for the esterase, with corncob giving the best ferulic acid yield. The corncob was incubated with T. fusca xylanase (Tfx) for 12h and then with the AXE esterase for an additional 12h. Ferulic acid accumulated to 396μM in the culture broth, a higher concentration than with esterase alone or with Tfx and esterase together for 24h.

Biological characterization of two Bacillus thuringiensis strains toxic against Spodoptera frugiperda

Two Bacillus thuringiensis strains isolated from diseased Spodoptera frugiperda larvae collected in the northwest of Argentina were molecularly and phenotypically characterized. Insecticidal activity against Spodoptera frugiperda larvae was also determined. Both strains were highly toxic against first instar larvae. One strain (Bacillus thuringiensis LSM) was found to be even more toxic than the reference strain Bacillus thuringiensis var. kurstaki 4D1. This strong biological effect was represented by both a higher mortality which reached 90%, and a shorter LT50. Molecular characterization showed that Bacillus thuringiensis LSM carried a cry gene profile identical to that of Bacillus thuringiensis var. kurstaki 4D1. Evaluation of length polymorphism of the intergenic transcribed spacers between the 16S and 23S rDNA genes revealed an identical pattern between native strains and Bacillus thuringiensis var. kurstaki 4D1. In contrast, phenotypic characterization allowed differentiation among the isolates by means of their extracellular esterase profiles. Lytic activity that would contribute to Bacillus thuringiensis effectiveness was also studied in both strains. Analyses like those presented in the current study are essential to identify the most toxic strains and to allow the exploitation of local biodiversity for its application in biological control programmes.

Degradation of poly (lactic acid) and nanocomposites by Bacillus licheniformis

The disposal problem due to non-degradable petroleum-based plastics has raised the demand for biodegradable polymers. The degradation of poly (lactic acid) (PLA) has been studied for several years, but the understanding of involved mechanisms is still incomplete. Based on our previous studies, and it is hypothesized an enzymatic involvement, the aim of this study was to continue investigations on the degradation of PLA and its nanocomposites by Bacillus licheniformis. Biodegradation of PLA and its nanocomposites (CLOISITE 30B and SOMASIF MEE) was performed on compression-molded, 25 × 25 × 0.6-mm films. Firstly, two plastic films were dipped into sterile nutrient broth inoculated with B. licheniformis and incubated at 32°C. Then, to verify if biodegradation was due to extracellular esterase, the culture broth was filtered to remove B. licheniformis cells, and the plastic materials were put into this broth. PLA degradation by B. licheniformis was accelerated by the presence of organoclays. After 5months in liquid culture, nanocomposites showed only the 10% of residual mass, compared with the 60% of pure PLA. Extracellular esterase activity was detected in the filtered culture broth confirming that PLA biodegradation was probably due to this enzyme action.

The Use of an Extracellular Ferulic Acid Esterase from Lactobacillus acidophilus K1 for the Release of Phenolic Acids During Mashing

A purified extracellular ferulic acid esterase from Lactobacillus acidophilus K1 was successfully used during mashing for the release of free phenolic acids into sweet wort. The enzyme was produced in bioreactors and partially purified to obtain the monoenzyme preparation. Release of free ferulic and vanillic acid into the wort at 52°C (with the use of 4.09-14.60 enzyme activity units/L of the mash) and ferulic acid at 62°C (14.60 units/L) was observed. Free p-OH-benzoic and syringic acids were effectively released at 26°C at each enzyme concentration used. Free p-OH-benzoic acid was also released by the enzyme (14.60 U/L) at 52°C-74°C. Free protocatechuic acid was effectively hydrolyzed by the enzyme preparation (8.75 U/L and 14.60 U/L) at 26°C-52°C. Free caffeic acid (effectively released at 26°C-62°C) originated from chlorogenic acid. No p-coumaric acid was released due to the action of bacterial esterase during mashing. Ferulic acid esterase from L. acidophilus K1 exhibited no ability to release free phenolic acids during mashing at 62°C or at 74°C due to its low thermostability. In conclusion, L. acidophilus K1 is an attractive source for the production of ferulic acid esterase, which can be useful for the release of antioxidant phenolic acids in the early stages of mashing.

Purification of a fungal cutinase by adsorptive bubble separation: A statistical approach

Abstract Nowadays, the use of enzymes for the pharmaceutical, cosmetic or food industry steadily increases. Although the specification and production of enzymes are steadily enlarged, the downstream processing strategies lag behind. Thus, the development or improvement of enzyme purification methods to efficient and competitive techniques is a main issue. Adsorptive bubble separation e.g. foam fractionation represents such a improvable separation process. In this study, foam fractionation was used to separate an extracellular esterase from submerged cultures of the basidiomycete Coprinopsis cinerea. Parameters such as pH-value and column design affect the transport of active enzymes into the foam phase and thus the efficiency of the foam fractionation process. The influence and interactions of various parameters regarding the performance of the foam fractionation process were analyzed using Design of Experiments (DoE). The use of Design of Experiments allowed a fast identification of impact parameters and a direct optimization of the foam fractionation process.

Purification and characterization of novel extracellular cholesterol esterase from Acinetobacter sp.

CHE4-1, a bacterial strain that belongs to the genus Acinetobacter and expresses high level of inducible extracellular cholesterol esterase (CHE), was isolated from feces of carnivore Panthera pardus var. The cholesterol esterase of the strain CHE4-1 was purified by ultrafiltration followed with DEAE-Sepharose FF chromatography and Phenyl-Sepharose CL-4B chromatography, and then by Sephadex G-50 gel filtration. Different from other known microbial cholesterol esterase, the purified CHE from CHE4-1 strain is a monomer with molecular weight of 6.5 kD and has high activity to both long-chain and short-chain cholesterol ester. Enzymatic activity was enhanced in the presence of metal ion Ca(2+), Zn(2+) and boracic acid, and was not significantly affected by several detergents including sodium cholate, Triton X100 and Tween-80. The enzyme was found to be stable during long-term aqueous storage at 4 °C, indicating its potential as a clinical diagnostic reagent. To the best of our knowledge, this is the first report regarding purification and characterization of CHE from Acinetobacter sp. The results demonstrated that this particular CHE is a novel cholesterol esterase.

Breeding and identification of novel koji molds with high activity of acid protease by genome recombination between Aspergillus oryzae and Aspergillus niger

Acid protease is essential for degradation of proteins during soy sauce fermentation. To breed more suitable koji molds with high activity of acid protease, interspecific genome recombination between A. oryzae and A. niger was performed. Through stabilization with d-camphor and haploidization with benomyl, several stable fusants with higher activity of acid protease were obtained, showing different degrees of improvement in acid protease activity compared with the parental strain A. oryzae. In addition, analyses of mycelial morphology, expression profiles of extracellular proteins, esterase isoenzyme profiles, and random amplified polymorphic DNA (RAPD) were applied to identify the fusants through their phenotypic and genetic relationships. Morphology analysis of the mycelial shape of fusants indicated a phenotype intermediate between A. oryzae and A. niger. The profiles of extracellular proteins and esterase isoenzyme electrophoresis showed the occurrence of genome recombination during or after protoplast fusion. The dendrogram constructed from RAPD data revealed great heterogeneity, and genetic dissimilarity indices showed there were considerable differences between the fusants and their parental strains. This investigation suggests that genome recombination is a powerful tool for improvement of food-grade industrial strains. Furthermore, the presented strain improvement procedure will be applicable for widespread use for other industrial strains.

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.

Extracellular Carbohydrate Esterase from the BasidiomyceteCoprinopsis cinereaReleased Ferulic and Acetic Acids from Xylan

cDNA encoding an extracellular carbohydrate esterase (CcEst1) was cloned from the basidiomycete Coprinopsis cinerea. The recombinant CcEst1 expressed in Pichia pastoris acted on p-nitrophenyl acetate, alpha-naphthyl acetate, and methyl hydroxycinnamic acids, except for methyl sinapic acid. The enzyme released ferulic and acetic acids from wheat arabinoxylan and acetylated xylan respectively. Activity increased on the addition of endo-beta-1,4-xylanase.