Production and characterization of esterase and lipase from <Emphasis Type="Italic">Haloarcula marismortui</Emphasis>

The present study was conducted to investigate the capability of Haloarcula marismortui to synthesize esterases and lipases, and the effect of physicochemical conditions on the growth and the production of esterases and lipases. Finally, the effect of NaCl concentration and temperature on esterase and lipase activities was studied using intracellular crude extracts. In order to confirm the genomic prediction about the esterase and lipase synthesis, H. marismortui was cultured on a rich medium and the crude extracts (intra- or extracellular) obtained were assayed for both activities using p-nitrophenyl esters and triacylglycerides as substrates. Studies on the kinetics of growth and production of esterase and lipase of H. marismortui were performed, reaching a maximum growth rate of 0.053 h⁻¹ and maximal productions of intracellular esterase and lipase of 2.094 and 0.722 U l⁻¹ using p-nitrophenyl valerate and p-nitrophenyl laurate, respectively. Both enzymes were produced as growth-associated metabolites. The effects of temperature, pH, and NaCl concentration on the growth rate and production of enzymes were studied by using a Box–Behnken response surface design. The three response variables were significantly influenced by the physicochemical factors and an interaction effect between temperature and NaCl concentration was also evidenced. The surface response method estimated the following maximal values for growth rate and productions of esterase and lipase: 0.086 h⁻¹ (at 42.5°C, pH 7.4, and 3.6 mol l⁻¹ NaCl), 2.3 U l⁻¹ (at 50°C, pH 7.5, and 4.3 mol l⁻¹ NaCl), and 0.58 U l⁻¹ (at 50°C, pH 7.6, and 4.5 mol l⁻¹ NaCl), respectively. Esterases were active at different salt concentrations, showing two optimal activities (at 0.5 and 5 mol l⁻¹ NaCl), which suggested the presence of two different esterases. Interestingly, in the absence of salt, esterase retained 50% residual activity. Esterases and lipase activities were maximal at 45°C and inactive at 75°C. This study represents the first report evidencing the synthesis of esterase and lipase by H. marismortui.     

Isozyme Polymorphism of Esterases in the Genus Lanistes (Mollusca: Prosobranchiata) and Genetic Analysis of Populations

Esterases from the digestive gland of the snails Lanistes carinatusand Lanistes boltenicollected from four Egyptian governorates were extracted and analyzed using starch gel electrophoresis and five substrates. Twelve esterase bands were detected in both Lanistes species. The esterase bands were distributed in three main zones, which could be classified as acetylesterases, carboxylesterases, and cholinesterases. Depending on the substrate specificity, inhibition properties, and relative mobility of esterase bands, the three zones of esterase activity could be traced to eight genetic loci. Locality-specific loci were found. Inter- and intrapopulation variations are discussed. There is an absence of equilibria at all esterase loci in all populations studied, and a high proportion of genetic diversity in different esterase loci. The absence of interspecific variations proves that Lanistessnails in Egypt belong to one species.     

A novel, extremely alkaliphilic and cold-active esterase from Antarctic desert soil

A novel, cold-active and highly alkaliphilic esterase was isolated from an Antarctic desert soil metagenomic library by functional screening. The 1,044 bp gene sequence contained several conserved regions common to lipases/esterases, but lacked clear classification based on sequence analysis alone. Moderate (<40%) amino acid sequence similarity to known esterases was apparent (the closest neighbour being a hypothetical protein from Chitinophaga pinensis), despite phylogenetic distance to many of the lipolytic “families”. The enzyme functionally demonstrated activity towards shorter chain p-nitrophenyl esters with the optimal activity recorded towards p-nitrophenyl propionate (C3). The enzyme possessed an apparent T_opt at 20°C and a pH optimum at pH 11. Esterases possessing such extreme alkaliphily are rare and so this enzyme represents an intriguing novel locus in protein sequence space. A metagenomic approach has been shown, in this case, to yield an enzyme with quite different sequential/structural properties to known lipases. It serves as an excellent candidate for analysis of the molecular mechanisms responsible for both cold and alkaline activity and novel structure–function relationships of esterase activity.     

Purification and properties of a highly enantioselective l-menthyl acetate hydrolase from Burkholderia cepacia

A highly enantioselective l-menthyl acetate esterase was purified to homogeneity from Burkholderia cepacia ATCC 25416, with a recovery of 4.8% and a fold purification of 22.7. The molecular weight of the esterase was found to be 37 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The N-terminal amino acid sequence was “MGARTDA”, and there was no homology in contrast to other Burkholderia sp. esterases. This enzyme preferentially hydrolyzed short-chain fatty acid esters of menthol with high stereospecificity and high hydrolytic activity, while long-chain l-menthyl esters were poor substrates. Considered its substrate specificity and N-terminal sequence, this esterase was concluded as a new enzyme belonging to the carboxylesterase group (EC 3.1.1.1) of esterase family. The optimum temperature and pH for enzyme activity using racemic menthyl acetate as substrate were 30 °C and 7.0, respectively. The esterase was more stable in the pH range of 7.0–9.0 and temperature range of 30–40 °C. Hydrolytic activity was enhanced by Ca²⁺, K⁺ and Mg²⁺, but completely inhibited by Hg²⁺, Cu²⁺, ionic detergents and phenylmethylsulfonyl fluoride (PMSF) at 0.01 M concentration.     

Characterization of a novel alkaline esterase from Altererythrobacter epoxidivorans CGMCC 1.7731T

A novel esterase gene (e25) was identified from Altererythrobacter epoxidivorans CGMCC 1.7731^T by genome sequence screening. The e25 gene is 948 nucleotides in length and encodes a 315?amino acid protein (E25) with a predicted molecular mass of 33,683 Da. A phylogenetic tree revealed that E25 belongs to the hormone-sensitive lipase (HSL) family of lipolytic enzymes. An activity assay of E25 showed that it exhibited the highest catalytic efficiency when using p-nitrophenyl caproate (C6) as a substrate. The optimum pH and temperature were determined to be approximately pH 9 and 45°C, and the K_m and V_max values were 0.12?mM and 1,772?µmol/min/mg, respectively. After an incubation at 40°C for 80?min, E25 retained 75% of its basal activity. The enzyme exhibited good tolerance to metal cations, such as Ba²⁺, Ca²⁺, and Cu²⁺ (10?mM), but its activity was strongly inhibited by Co²⁺, Ni²⁺, Mn²⁺, and Zn²⁺. The E25 enzyme was stimulated by glycerol and retained over 60% of its basal activity in the presence of 1% Tween-80 and Triton X-100. Overall, the activity of E25 under alkaline conditions and its organic solvent and detergent tolerance indicate that E25 could be useful as a novel industrial catalyst in biotechnological applications.     

Chitinase production by <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> and <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>: Their potential in antifungal biocontrol

Thirty bacterial strains were isolated from the rhizosphere of plants collected from Egypt and screened for production of chitinase enzymes. Bacillus thuringiensis NM101-19 and Bacillus licheniformis NM120-17 had the highest chitinolytic activities amongst those investigated. The production of chitinase by B. thuringiensis and B. licheniformis was optimized using colloidal chitin medium amended with 1.5% colloidal chitin, with casein as a nitrogen source, at 30°C after five days of incubation. An enhancement of chitinase production by the two species was observed by addition of sugar substances and dried fungal mats to the colloidal chitin media. The optimal conditions for chitinase activity by B. thuringiensis and B. licheniformis were at 40°C, pH 7.0 and pH 8.0, respectively. Na⁺, Mg²⁺, Cu²⁺, and Ca²⁺ caused enhancement of enzyme activities whereas they were markedly inhibited by Zn²⁺, Hg²⁺, and Ag⁺. In vitro, B. thuringiensis and B. licheniformis chitinases had potential for cell wall lysis of many phytopathogenic fungi tested. The addition of B. thuringiensis chitinase was more effective than that of B. licheniformis in increasing the germination of soybean seeds infected with various phytopathogenic fungi.     

Mining bacterial genomes for novel arylesterase activity

One hundred and seventy-one genes encoding potential esterases from 11 bacterial genomes were cloned and overexpressed in Escherichia coli; 74 of the clones produced soluble proteins. All 74 soluble proteins were purified and screened for esterase activity; 36 proteins showed carboxyl esterase activity on short-chain esters, 17 demonstrated arylesterase activity, while 38 proteins did not exhibit any activity towards the test substrates. Esterases from Rhodopseudomonas palustris (RpEST-1, RpEST-2 and RpEST-3), Pseudomonas putida (PpEST-1, PpEST-2 and PpEST-3), Pseudomonas aeruginosa (PaEST-1) and Streptomyces avermitilis (SavEST-1) were selected for detailed biochemical characterization. All of the enzymes showed optimal activity at neutral or alkaline pH, and the half-life of each enzyme at 50°C ranged from < 5 min to over 5 h. PpEST-3, RpEST-1 and RpEST-2 demonstrated the highest specific activity with pNP-esters; these enzymes were also among the most stable at 50°C and in the presence of detergents, polar and non-polar organic solvents, and imidazolium ionic liquids. Accordingly, these enzymes are particularly interesting targets for subsequent application trials. Finally, biochemical and bioinformatic analyses were compared to reveal sequence features that could be correlated to enzymes with arylesterase activity, facilitating subsequent searches for new esterases in microbial genome sequences.     

Molecular cloning and characterization of a bifunctional xylanolytic enzyme from Neocallimastix patriciarum

A cDNA encoding a bifunctional acetylxylan esterase/xylanase, XynS20E, was cloned from the ruminal fungus Neocallimastix patriciarum. A putative conserved domain of carbohydrate esterase family 1 was observed at the N-terminus and a putative conserved domain of glycosyl hydrolase family 11 was detected at the C-terminus of XynS20E. To examine the enzyme activities, XynS20E was expressed in Escherichia coli as a recombinant His₆ fusion protein and purified by immobilized metal ion-affinity chromatography. Response surface modeling combined with central composite design and regression analysis was then applied to determine the optimal temperature and pH conditions of the recombinant XynS20E. The optimal conditions for the highest xylanase activity of the recombinant XynS20E were observed at a temperature of 49°C and a pH of 5.8, while those for the highest carbohydrate esterase activity were observed at a temperature of 58°C and a pH of 8.2. Under the optimal conditions for the enzyme activity, the xylanase and acetylxylan esterase specific activities of the recombinant XynS20E toward birchwood xylan were 128.7 and 873.1 U mg⁻¹, respectively. To our knowledge, this is the first report of a bifunctional xylanolytic enzyme with acetylxylan esterase and xylanase activities from rumen fungus.     

Es-6, a further polymorphic esterase in the rat

A further polymorphic rat esterase with broad tissue expression and restricted substrate specificity is described and tentatively called Es-6. Inbred rat strains have either fixed allele Es-6^F or fixed allele Es-6^S. Es-6 is not linked to the established esterase cluster consisting of the eight esterase loci Es-1, Es-2, Es-3M, Es-4M, Es-4W, Es-5 (=Es-3W), Es-7, and Es-8 in LG V of the rat or to RT1, Gc, c, a, and h. Esterases with apparently identical biochemical and genetical characteristics are Es-17 of the mouse and Es-A4 of humans.Supported by the Deutsche Forschungsgemeinschaft (Be 352/13 and Gu 105).     

Production and Properties of Lipase from a Newly Isolated Chromobacterium

Out of some 750 strains of microorganisms, a potent bacterium for lipase production was isolated from soil and was identified as Chromobacterium viscosum.

The bacterium accumulates lipase in culture fluid when grown aerobically at 26°C for 3 days in a medium composed of soluble starch, soy bean meal, lard and inorganic salts.

Chromobacterium lipase had an optimum pH of 7.0 for activity at 37°C, and an optimal temperature of 65°C at pH 7.0. The enzyme retained 80% of the activity when heated for 10 min at 70°C. This lipase was capable of hydrolyzing a variety of natural fats and oils, and it was more active on lard and butter than on olive oil. The activity was stimulated by Ca²⁺, Mg²⁺, Mn²⁺ and inhibited by Cu²⁺, Hg²⁺ and Sn²⁺. It was not diminished but rather stimulated by a high concentration of bile-salts.     

Extremely thermostable esterases from the thermoacidophilic euryarchaeon Picrophilus torridus

Two genes encoding esterases EstA and EstB of Picrophilus torridus were identified by the means of genome analysis and were subsequently cloned in Escherichia coli. PTO 0988, which is encoding EstA, consists of 579 bp, whereas PTO 1141, encoding EstB, is composed of 696 bp, corresponding to 192 aa and 231 aa, respectively. Sequence comparison revealed that both biocatalysts have low sequence identities (14 and 16%) compared to previously characterized enzymes. Detailed analysis suggests that EstA and EstB are the first esterases from thermoacidophiles not classified as members of the HSL family. Furthermore, the subunits with an apparent molecular mass of 22 and 27 kDa of the homotrimeric EstA and EstB, respectively, represent the smallest esterase subunits from thermophilic microorganisms reported to date. The recombinant esterases were purified by Ni²⁺ affinity chromatography, and the activity of the purified esterases was measured over a wide pH (pH 4.5–8.5) and temperature range (10–90°C). Highest activity of the esterases was measured at 70°C (EstA) and 55°C (EstB) with short pNP-esters as preferred substrates. In addition, esters of the non-steroidal anti-inflammatory drugs naproxen, ketoprofen, and ibuprofen are hydrolyzed by both EstA and EstB. Extreme thermostability was measured for both enzymes at temperatures as high as 90°C. The determined half-life (t _1/2) at 90°C was 21 and 10 h for EstA and EstB, respectively. Remarkable preservation of esterase activity in the presence of detergents, urea, and commonly used organic solvents complete the exceptional phenotype of EstA and EstB.     

Isolation and characterization of a family VII esterase derived from alluvial soil metagenomic library

A novel esterase gene, estDL30, was isolated from an alluvial metagenomic library using function-driven screening. estDL30 consisted of 1,524 nucleotides and encoded a 507-amino acid protein. Sequence analysis revealed that EstDL30 is similar to many type B carboxylesterases, containing a G-E-S-A-G pentapeptide with a catalytic Ser residue. Phylogenetic analysis suggested that EstDL30 belongs to the family VII lipases, together with esterases from Bacillus subtilis (P37967), Streptomyces coelicolor A3(2) (CAA22794), and Arthrobacter oxydans (Q01470). Purified EstDL30 showed its highest catalytic efficiency toward p-nitrophenyl butyrate, with a k _cat of 2293 s⁻¹ and k _cat/K _m of 176.4 s⁻¹mM⁻¹; however, little activity was detected when the acyl chain length exceeded C₈. Biochemical characterization of EstDL30 revealed that it is an alkaline esterase that possesses maximal activity at pH 8 and 40° C. The effects of denaturants and divalent cations were also investigated. EstDL30 tolerated well the presence of methanol and Tween 20. Its activity was strongly inhibited by 1 mM Cu²⁺ and Zn²⁺, but stimulated by Fe²⁺. The unique properties of EstDL30, its high activity under alkaline conditions and stability in the presence of organic solvents, may render it applicable to organic synthesis.     

Morphological,physiological, and molecular characterization of actinomycetes isolated from dry soil,rocks, and monument surfaces

In an extended study on the biodiversity of rock-dwelling bacteria, the colony and cell morphology, physiology, protein patterns, and 16S rDNA sequences of 17 bacterial strains isolated from different surfaces of rocks, stones, and monuments and from various geographical locations were characterized. All except one strain, which was found to be a Bacillus, were members of the order Actinomycetales. The majority of the strains either were closely related to Geodermatophilus obscurus, which was also analyzed in this study, or formed a closely related sister taxon. All of these strains were isolated from the surface of marble in Namibia and Greece and from limestone from the Negev desert, Israel. One strain, G10, of Namibia origin was equidistantly related to Geodermatophilus obscurus, Frankia alni, Sporichthya polymorpha, and Acidothermus cellulolyticus. Three strains from rock varnish in the Mojave desert, California, were found to be highly related to Arthrobacter (formerly Micrococcus) agilis. All clusters could be confirmed from results of studies on morphological and physiological properties and from banding patterns of whole cell proteins. Based on the results of tests, four additional strains were assigned to the lineage defined by strain G10. Received: 16 October 1995 / Accepted: 4 April 1996     

Purification and characterization of an intracellular esterase from a Fusarium species capable of degrading dimethyl terephthalate

Esterase is the key enzyme involved in microbial degradation of phthalate esters (PAEs). In this study, an intracellular esterase was purified from a coastal sediment fungus Fusarium sp. DMT-5-3 capable of utilizing dimethyl terephthalate (DMT) as a substrate. The purified enzyme is a polymeric protein consisting of two identical subunits with a molecular mass of about 84 kDa. The enzyme showed a maximum esterase activity at 50 °C and was stable below 30 °C. The optimal pH was 8.0 and the enzyme was stable between pH 6.0 and 10.0. The esterase activity was inhibited by Cr³⁺, Hg²⁺, Cu²⁺, Zn²⁺, Ni²⁺, and Cd²⁺. Substrate specificity analysis showed that the enzyme was specific to DMT hydrolysis, but had no effect on other isomers of dimethyl phthalate esters (DMPEs) or monomethyl phthalate esters (MMPEs). These findings suggest that the phthalate esterase produced by Fusarium sp. DMT-5-3 is inducible and distinctive esterases involved in hydrolysis of the two carboxylic ester linkages of DMPEs.     

Molecular cloning,purification, and characterization of a novel thermostable cinnamoyl esterase from Lactobacillus helveticus KCCM 11223

A gene encoding cinnamoyl esterase (CE), which breaks down chlorogenic acid (ChA) into caffeic and quinic acids, was cloned from Lactobacillus helveticus KCCM 11223. The gene with an open reading frame of 759 nucleotides was expressed in Escherichia coli, which resulted in a 51.6-fold increase in specific activity compared to L. helveticus KCCM 11223. The recombinant CE exists as a monomeric enzyme having a molecular weight of 27.4?kDa. Although the highest activity was observed at pH 7, the enzyme showed stable activity at pH 4.0–10.0. Its optimum temperature was 65°C, and it also possessed a thermophilic activity: the half-life of CE was 24.4?min at 65°C. The half-life of CE was 145.5, 80.5, and 24.4?min at 60, 62, and 65°C, respectively. The K_m and V_max values for ChA were 0.153?mM and 559.6?µM/min, respectively. Moreover, the CE showed the highest substrate specificity with methyl caffeate among other methyl esters of hydroxycinnamic acids such as methyl ferulate, methyl sinapinate, methyl p-coumarate, and methyl caffeate. Ca²⁺, Cu²⁺, and Fe²⁺ significantly reduced the relative activity on ChA up to 70%. This is the first report on a thermostable CE from lactic acid bacteria that can be useful to hydrolyze ChA from plant cell walls.     

Purification and biochemical characterization of feruloyl esterases from Aspergillus terreus MTCC 11096

Aspergillus terreus MTCC 11096 isolated from the soils of agricultural fields cultivating sweet sorghum was previously identified to produce feruloyl esterases (FAEs). The enzymes responsible for feruloyl esterase activity were purified to homogeneity and named as AtFAE‐1, AtFAE‐2, and AtFAE‐3. The enzymes were monomeric having molecular masses of 74, 23 and 36 kDa, respectively. Active protein bands were identified by a developed pH‐dependent zymogram on native PAGE. The three enzymes exhibited variation in pH tolerance ranging between pH 5–8 and thermostability of up to 55°C. Inhibition studies revealed that the serine residue was essential for feruloyl esterase activity; moreover aspartyl and glutamyl residues are not totally involved at the active site. Metal ions such as Ca²⁺, K⁺, and Mg²⁺ stabilized the enzyme activity for all three FAEs. Kinetic data indicated that all three enzymes showed catalytic efficiencies (k_cat/K_m) against different synthesized alkyl and aryl esters indicating their broad substrate specificity. The peptide mass fingerprinting by MALDI/TOF‐MS analysis and enzyme affinity toward methoxy and hydroxy substituents on the benzene ring revealed that the AtFAE‐1 belonged to type A while AtFAE‐2 and AtFAE‐3 were type C FAE. The FAEs could release 65 to 90% of ferulic acid from agrowaste substrates in the presence of xylanase. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:924–932, 2013     

Polynucleotide Phosphorylase from Acromobacter sp. KR 170-4

Eighty-five strains of bacteria were screened for selection of microorganisms suitable for industrial production of polynucleotides. Among these bacteria, Achromobacter sp. KR 170-4 (ATCC 21942) was found to be rich in polynucleotide Phosphorylase (PNPase) in its “salt-shockate” as compared with the other strains tested. PNPase was purified about 50-fold from the “salt-shockate” of Achromobacter sp. KR 170-4, and properties of the enzyme were elucidated. Optimal pH for reaction was 10.1. Stable pH range at 37°C was between pH 6.5 and 10.5. Optimal temperatures were 46°C for polymerization of ADP or IDP, and 43°C for CDP or UDP. The enzyme was stable below 55°C at pH 9.2. The enzyme required Mn²⁺ rather than Mg²⁺ unlike the other PNPases reported. Optimal concentration of Mn²⁺ was 6 mM.     

Purification and characterization of cyclodextrin β-glucanotransferase from novel alkalophilic bacilli

The discovery of novel bacterial cyclodextrin glucanotransferase (CGTase) enzyme could provide advantages in terms of its production and relative activity. In this study, eight bacterial strains isolated from soils of a biodiversity-rich vegetation in Egypt based on their hydrolyzing activity of starch, were screened for CGTase activity, where the most active strain was identified as Bacillus lehensis. Optimization process revealed that the using of rice starch (25 %) and a mixture of peptone/yeast extract (1 %) at pH 10.5 and 37 °C for 24 h improved the bacterial growth and enzyme activity. The bacterial CGTase was successively purified by acetone precipitation, gel filtration chromatography in a Sephadex G-100 column and ion exchange chromatography in a DEAE-cellulose column. The specific activity of the CGTase was increased approximately 274-fold, from 0.21 U/mg protein in crude broth to 57.7 U/mg protein after applying the DEAE-cellulose column chromatography. SDS-PAGE showed that the purified CGTase was homogeneous with a molecular weight of 74.1 kDa. Characterization of the enzyme exhibited optimum pH and temperature of 7 and 60 °C, respectively. CGTase relative activity was strongly inhibited by Mg²⁺, Zn²⁺, Al³⁺ and K⁺, while it was slightly enhanced by 5 and 9 % with Cu²⁺ and Fe²⁺ metal ions, respectively.     

Improved expression of a soluble single chain antibody fusion protein containing tumor necrosis factor in <Emphasis Type="Italic">Escherichia coli</Emphasis>

An epoxide hydrolase gene of about 0.8 kb was cloned from Rhodococcus opacus ML-0004, and the open reading frame (ORF) sequence predicted a protein of 253 amino acids with a molecular mass of about 28 kDa. An expression plasmid carrying the gene under the control of the tac promotor was introduced into Escherichia coli, and the epoxide hydrolase gene was successfully expressed in the recombinant strains. Some characteristics of purified recombinant epoxide hydrolase were also studied. Epoxide hydrolase showed a high stereospecificity for l(+)-tartaric acid, but not for d(+)-tartaric acid. The epoxide hydrolase activity could be assayed at the pH ranging from 3.5 to 10.0, and its maximum activity was obtained between pH 7.0 and 7.5. The enzyme was sensitive to heat, decreasing slowly between 30°C and 40°C, and significantly at 45°C. The enzyme activity was activated by Ca²⁺ and Fe²⁺, while strongly inhibited by Ag⁺ and Hg⁺, and slightly inhibited by Cu²⁺, Zn²⁺, Ba²⁺, Ni⁺, EDTA–Na₂ and fumarate.     

Electrophoretic patterns of esterases and lactate-and l-malate-dehydrogenases from Flavobacterium species

Esterases and lactate-(LDH) and l-malate-(MDH) dehydrogenases of 64 strains of 13 species of Flavobacterium (particularly 46 strains of F. meningosepticum belonging to 15 different serotypes) were analyzed by horizontal polyacrylamide-agarose gel electrophoresis. Twenty-five esterase types were detected; these enzymes were distinguished by their spectra of hydrolytic activity towards five synthetic substrates (hydrolytic type) and their electrolytic mobilities (electrophoretic type). No variant of LDH was detected in all the strains of Flavobacterium. No variant of MDH was detected in three species: F. aquatile, F. branchiophilum, and F. breve and in serotype I, J and L. of F. meningosepticum. These results within the genus Flavobacterium permit precise identification, which may be useful for characterization of strains kept in collections as well as for epidemiological studies.