P-nitrophenyl Esters Research Articles

Molecular Characterization of a Novel Arylesterase from the Wine-Associated Acetic Acid Bacterium Gluconobacter oxidans 621H

An arylesterase from the wine-making acetic acid bacterium, Gluconobacter oxidans, was cloned and expressed into Escherichia coli. The soluble 76.8 kDa dimeric enzyme obtained, Est0881, was purified in only two steps with a 3.1-fold purification, 43% recovery, and a specific activity of 214 U/mg for the hydrolysis of p-nitrophenyl acetate. The optimum pH and temperature were 7.0 and 40 °C, respectively. The substrate specificity of this arylesterase was higher toward short chain p-nitrophenyl esters (C(2) to C(4)) and also toward aromatic esters, such as phenyl acetate. The deduced amino acid sequence shares high identity with esterases of the HSL family. The inhibition results obtained showed that the enzyme was a serine esterase, belonging to the A-esterases (arylesterases) and contains a catalytic triad composed of Ser163, Asp263, and His293 in the active site. Est0881 retained significant activity under conditions simulating those of wine-making (75% activity at 20% ethanol), making it a promising biocatalyst for modulating the final aroma of wine.

Micellization and Catalytic Properties of Cationic Surfactants with Head Groups Functionalized with a Hydroxyalkyl Fragment

The catalytic activity of two homological series of cationic surfactants bearing a hydroxyalkyl fragment in the head groups R(CH3)2N+(CH2CH2OH)Br– and R(CH3)2N+(CH2CH2CH2OH)Br– toward the cleavage of the p-nitrophenyl esters of carbonic acids of different hydrophobicity (acetate, caprilate, caprinate, laurate, myristate) is shown to exceed that of typical cationic surfactants with the trimethylammonium (TMA) headgroup. The catalytic effect increases with the alkyl chain length of surfactants and nonmonotonous changes in the series: acetate < caprilate < myristate < laurate < caprinate, reaching 750 times. NMR and IR spectroscopy studies and the surface potential calculations revealed that the higher catalytic effect of hydroxyalkylated surfactants is not due to their higher surface potential and binding capacity toward substrates. This is in line with finding that binding constants for TMA series are higher than for their hydroxyalkylated analogues, which was demonstrated by the fitting of kinetic data in...

Gene Cloning and Characterization of a Cold-Adapted Esterase from Acinetobacter venetianus V28

Acinetobacter venetians V28 was isolated from the intestine of righteye flounder, Poecilopsetta plinthus caught in Vietnam seawater, and the esterase gene was cloned using a shotgun method. The amino acid sequence deduced from the nucleotide sequence (1,017 bp) corresponded to a protein of 338 amino acid residues with a molecular weight of 37,186. The esterase had 87% and 72% identities with the lipases of A. junii SH205 and A. calcoaceticus RUH2202, respectively. The esterase contained a putative leader sequence, as well as the conserved catalytic triad (Ser, His, Asp), consensus pentapeptide GXSXG, and oxyanion hole sequence (HG). The protein from the strain V28 was produced in both a soluble and an insoluble form when the Escherichia coli cells harboring the gene were cultured at 18 degrees C. The maximal activity of the purified enzyme was observed at a temperature of 40 degrees C and pH 9.0 using p-NP-caprylate as substrate; however, relative activity still reached to 70% even at 5 degrees C with an activation energy of 3.36 kcal/mol, which indicated that it was a cold-adapted enzyme. The enzyme was a nonmetalloprotein and was active against p-nitrophenyl esters of C4, C8, and C14. Remarkably, this enzyme retained much of its activity in the presence of commercial detergents and organic solvents. This cold-adapted esterase will be applicable as catalysts for reaction in the presence of organic solvents and detergents.

Expression and Characterization of a Novel Enantioselective Lipase from Aspergillus fumigatus

A 1,080-bp cDNA (CGMCC 2873) encoding of a cold-active lipase of Aspergillus fumigatus (AFL67) was cloned and expressed in Escherichia coli for the first time. The new lipase, AFL67, was one-step purified by 8.30 folds through Ni-NTA affinity chromatography with a recovery of 86.8 %. The specific activity of purified AFL67 was 449 U mg(-1) on p-NP hexanoate. AFL67 preferentially hydrolyzed p-nitrophenyl esters of short- and medium-chain fatty acids, with p-nitrophenyl hexanoate the maximum. The optimum temperature and pH was 15 °C and 7.5, respectively. The purified AFL67 was stable at 10-25 °C for 30 min, and in the pH range of 6.0-9.0 for 16 h (at 4 °C). Its activity was increased by 47 and 50 %, in the presence of 10 % (v/v) ethanol and isopropanol, respectively. The new lipase AFL67 highly enantioselectively deacylated (S)-α-acetoxyphenylacetic acid (APA) and o-Cl-APA, m-Cl-APA, and p-Cl-APA to (S)-mandelic acid and its derivates. These features render this cold-active novel lipase AFL67 attractive for biotechnological applications in the field of enantioselective synthesis of chiral mandelic acids, o-acylated mandelic acids, and their derivates and detergent additives.

Computational Design of Catalytic Dyads and Oxyanion Holes for Ester Hydrolysis

Nucleophilic catalysis is a general strategy for accelerating ester and amide hydrolysis. In natural active sites, nucleophilic elements such as catalytic dyads and triads are usually paired with oxyanion holes for substrate activation, but it is difficult to parse out the independent contributions of these elements or to understand how they emerged in the course of evolution. Here we explore the minimal requirements for esterase activity by computationally designing artificial catalysts using catalytic dyads and oxyanion holes. We found much higher success rates using designed oxyanion holes formed by backbone NH groups rather than by side chains or bridging water molecules and obtained four active designs in different scaffolds by combining this motif with a Cys-His dyad. Following active site optimization, the most active of the variants exhibited a catalytic efficiency (k(cat)/K(M)) of 400 M(-1) s(-1) for the cleavage of a p-nitrophenyl ester. Kinetic experiments indicate that the active site cysteines are rapidly acylated as programmed by design, but the subsequent slow hydrolysis of the acyl-enzyme intermediate limits overall catalytic efficiency. Moreover, the Cys-His dyads are not properly formed in crystal structures of the designed enzymes. These results highlight the challenges that computational design must overcome to achieve high levels of activity.

Specific Enzyme-Catalyzed Hydrolysis and Synthesis in Aqueous and Organic Medium Using Biocatalysts with Lipase Activity from Aspergillus niger MYA 135

In the present study, the specific hydrolytic activity of three biocatalysts such as the constitutive mycelium-bound lipase, the induced mycelium-bound lipase and the lyophilized induced supernatant from A. niger MYA 135 was evaluated in both aqueous and organic media. A direct correlation between activity in water and n-hexane was not observed for the same hydrolytic reaction. The n-hexane/water activity ratio (R O/A) was applied to characterize the activity in organic medium. The three biocatalysts showed R O/A values higher than 1 for hydrolysis of long-chain fatty acid esters, demonstrating a higher specific hydrolytic activity in organic solvent than in water. A different behavior was observed during hydrolysis of middle-chain fatty acid esters, which was higher in aqueous medium (R O/A < 1). Transesterifications of different alcohols with various p-nitrophenyl derivatives using all three biocatalysts preparations were also evaluated in n-hexane. For methanolysis and ethanolysis, the constitutive mycelium-bound lipase displayed an interesting preference for C16 substrate (p-nitrophenyl palmitate). The induced mycelium-bound lipase showed high specific transesterification activities in the presence of water-miscible alcohols and middle-chain fatty acid esters (p-nitrophenyl caprate and p-nitrophenyl laurate), being the highest specific transesterification activity (91.4 ± 1.7 mU/gdw) observed in a reaction mixture containing propanol and p-nitrophenyl laurate. Finally, both p-nitrophenyl caprate (C10) and p-nitrophenyl laurate (C12) were preferentially methanolized by the lyophilized induced supernatant, being this lipase activity the most specific biocatalyst preparation under transesterification conditions. A selectivity-based analysis of each lipase preparation toward transesterification or hydrolysis in organic medium was evaluated as well.

Cloning, expression and characterization of a novel esterase from Bacillus pumilus

The gene encoding esterase (CE1) from Bacillus pumilus ARA with a calculated molecular weight of 28.4 kDa was cloned, sequenced and efficiently expressed in Escherichia coli. The open reading frame of 747 nucleotides encoded a protein, which was classified as a carboxylesterase with an identity of 87 % to esterase from Bacillus subtilis 168. Recombinant CE1 was purified in a single step to electrophoretic homogeneity by IMAC (Ni2+). The enzyme displayed maximum activity toward p-nitrophenyl (pNP) acetate at 37–40 °C and pH 6.5–7.0. It was stable in the pH range from 6.5 to 8.0, and at temperature from 25 to 40 °C. Among four p-nitrophenyl esters tested, the best substrate was pNP acetate with K m and k cat values of 0.33 mM and 4.07 s−1, respectively. Amounts of 2 mM Ca2+ and Co2+ significantly increased the esterase activity to 190 and 121 %, respectively. These results suggest that CE1 has very attractive applications of increasing feed digestibility in animal nutrition in this moderate temperature range.

Directed evolution of poly[(R)-3-hydroxybutyrate] depolymerase using cell surface display system: functional importance of asparagine at position 285

Poly[(R)-3-hydroxybutyrate] (PHB) depolymerase from Ralstonia pickettii T1 (PhaZRpiT1) consists of three functional domains to effectively degrade solid PHB materials, and its catalytic domain catalyzes the ester bond cleavage of the substrate. We performed the directed evolution of PhaZRpiT1 targeted at the catalytic domain in combination with the cell surface display method to effectively screen for mutants with improved p-nitrophenyl butyrate (pNPC4) activity. Mutated PhaZRpiT1 genes generated by error-prone PCR were fused to the oprI gene to display them as fusion proteins on Escherichia coli cell surface. Some cells displaying the mutant enzymes showed a two- to fourfold increase in pNPC4 hydrolysis activity relative to cells displaying wild-type enzyme. These mutant genes were recombined by a staggered extension process and the recombined enzymes were displayed to result in a five- to eightfold higher pNPC4 hydrolysis activity than the wild type. To further evaluate the mutation effects, unfused and undisplayed enzymes were prepared and applied to the hydrolysis of p-nitrophenyl esters having different chain lengths (pNPCn; n = 2-6) and PHB degradation. One specific second-generation mutant showed an approximately tenfold increase in maximum rate for pNPC3 hydrolysis, although its PHB degradation efficiency at 1 μg/mL of enzyme concentration was approximately 3.5-fold lower than that of the wild type. Gene analysis showed that N285D or N285Y mutations were found in six of the seven improved second-generation mutants, indicating that Asn285 probably participates in the regulation of substrate recognition and may be more favorable for PHB degradation process than other amino acid residues.

Use of bacteria for rapid, pH-neutral, hydrolysis of the model hydrophobic carboxylic acid ester p-nitrophenyl picolinate

Caulobacter crescentus, Escherichia coli and Bacillus subtilis cultures promote the hydrolysis of the model ester p-nitrophenyl picolinate (PNPP) at neutral pH with high efficiency. Hydrolysis is related to cell concentration, while the interaction of PNPP with both bacterial cells and their extracellular molecules is required for a maximum rate of PNPP hydrolysis in C. crescentus cultures. Furthermore, C. crescentus cultures hydrolyse PNPP at concentrations useful in synthetic chemistry.

Characterization of a New Cold-adapted Lipase from Pseudomonas sp. TK-3

A psychrotrophic Pseudomonas sp. TK-3 was isolated from dirty and cool stream water in Toyama, Japan from which we cloned and characterized the bacterial lipase LipTK-3. The sequenced DNA fragment contains an open reading frame of 1,428 bp that encoded a protein of 476 amino acids with an estimated molecular mass of 50,132 Da. The lipase showed high sequence similarity to those of subfamily Ι.3 lipase and had a conserved GXSXG motif around the catalytic Ser residue. Its optimal temperature was 20-25 °C, lower than in most other subfamily Ι.3 lipases. The lipase exhibited about 30 % of maximal activity at 5 °C. The optimal pH value was 8.0. The activity was strongly inhibited by EDTA and was highly dependent on Ca(2+). Tricaprylin and p-nitrophenyl caprylate were the most favorable substrates among the triglycerides and p-nitrophenyl esters, respectively. LipTK-3 also showed high activity towards natural substrates including edible vegetable oils and animal fats. Furthermore, LipTK-3 was very active and stable in the presence of several detergents, metal ions, and organic solvents. This cold-adapted lipase may prove useful for future applications.

The Isolation and Culture Characterization of a Lipolytic Enzyme Producing Strain from Meju

For screening of useful enzymes producing microorganisms from Meju, we isolated high lipase producing strains and their lipolytic enzyme activities were then tested. The lipolytic enzyme activities of isolated microorganisms were therefore tested on the Y124 strain. The gene sequence analysis of ITS from Y124 strain revealed Yarrowia lipolytica. Lipase production by the Y124 strain was studied in media containing various carbon sources. The Y124 strain drastically increased lipolytic enzyme activity in YPO media containing olive oil, as well as in YPDO media containing both olive oil and glucose. Maximal lipase production was achieved in YPD (yeast extract-peptone-D-glucose) media containing 0.7% olive oil when cultured at for 8 hrs. The lipase produced from the Y124 strain showed the highest activity in p-NPO (p-nitrophenyl octanoate ()), amongst the various p-nitrophenyl esters.

Purification and biochemical characterization of a cold-active lipase from Antarctic sea ice bacteria Pseudoalteromonas sp. NJ 70

An extracellular cold-active lipase from Antarctic sea ice bacteria Pseudoalteromonas sp. NJ 70 was purified and characterized. The overall purification based on lipase activity was 27.5-fold with a yield of 25.4 %. The purified lipase showed as a single band on SDS-PAGE with an apparent molecular weight of 37 kDa. The optimum temperature and pH were 35 °C and 7.0, respectively. The lipase activity was enhanced by Ca(2+) and Mg(2+), while was partially inhibited by other metals such as Cu(2+), Zn(2+), Ba(2+), Pb(2+), Fe(2+) and Mn(2+). The lipase had high tolerance to a wide range of NaCl concentrations (0-2 M NaCl). It exhibited high levels of activity in the presence of DTT, Thiourea, H(2)O(2) as well as in the presence of various detergents such as Span 60, Tween-80, Triton X-100. In addition, the lipase showed a preference for long-chain p-nitrophenyl esters (C(12)-C(18)). These results indicated that this lipase could be a novel cold-active lipase.

Self-Assembly of a Catalytic Multivalent Peptide–Nanoparticle Complex

Catalytically active peptide-nanoparticle complexes were obtained by assembling small peptide sequences on the surface of cationic self-assembled monolayers on gold nanoparticles. When bound to the surface, the peptides accelerate the transesterification of the p-nitrophenyl ester of N-carboxybenzylphenylalanine by more than 2 orders of magnitude. The gold nanoparticle serves as a multivalent scaffold for bringing the catalyst and substrate into close proximity but also creates a local microenvironment that further enhances the catalysis. The supramolecular nature of the ensemble permits the catalytic activity of the system to be modulated in situ.

Characterization of a novel esterase isolated from intertidal flat metagenome and its tertiary alcohols synthesis

Abstract A gene coding for an esterase (EstEH112) was isolated from metagenome originated from Korean intertidal flat sediment. The putative esterase gene encoded a 340 amino acids protein with characteristic residues of lipolytic enzymes such as a conserved pentapeptide (GXSXG), the typical catalytic S-D-H triad, and a GGG(A)X-motif in the oxyanion hole near the active site similar to the hormone sensitive lipase (HSL) family. p-Nitrophenyl butyrate was the best substrate for the enzyme among the other p-nitrophenyl esters investigated. The apparent optimal temperature and pH for EstEH112 was 35 °C and at pH 8.0, respectively. EstEH112 efficiently catalyzed the hydrolysis of various large tertiary alcohol esters. These characteristics of EstEH112 make it a potential candidate for application in biocatalysis.

Characterization of a novel extracellular lipase from a halophilic isolate, Chromohalobacter sp. LY7-8

A newly moderate halophilic strain LY7-8 with high lipolytic activity was isolated from salt lake of Yuncheng, China. Biochemical and physiological characterization, along with 16S rRNA sequence analysis placed the isolate in the genus Chromohalobacter. Lipase production started from the early-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 of C8 to C18, indicating a lipase activity. Enzyme purification was carried out by ammonium sulphate precipitation and Sephacryl S-100 gel filtration chromatography. Molecular weight of the lipase was determined to be 44 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme was highly active over broad temperature (30 to 90°C), pH (6.0-12.0) and NaCl concentration (0 to 20%) ranges, showing optimal activity at 60°C, pH 9.0 and 12.5% NaCl. Significant inhibition of the lipase was shown by ethylenediaminetetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF) and diethyl pyrocarbonate (DEPC), indicating it was a metalloenzyme with serine and histidine residues essential for its catalytic function. Moreover, it exhibited high activity in the presence of surfactants, such as SDS and Triton X-100. Results from the present study indicated that the extracellular lipase from LY7-8 may have considerable potential for industrial application from the perspectives of its properties. Key words: Moderately halophile, lipase, purification, Chromohalobacter

Cloning, expression and characterization of a halotolerant esterase from a marine bacterium Pelagibacterium halotolerans B2T

An esterase PE10 (279 aa) from Pelagibacterium halotolerans B2(T) was cloned and overexpressed in Escherichia coli Rosetta in a soluble form. The deduced protein was 29.91kDa and the phylogenetic analysis of the deduced amino acids sequence showed it represented a new family of lipolytic enzymes. The recombinant protein was purified by Ni-NTA affinity chromatography column and the characterization showed its optimal temperature and pH were 45°C and pH 7.5, respectively. Substrate specificity study showed PE10 preferred short chain p-nitrophenyl esters and exhibited maximum activity toward p-nitrophenyl acetate. In addition, PE10 was a halotolerant esterase as it was still active under 4M NaCl. Three-dimensional modeling of PE10 suggested that the high negative electrostatic potential on the surface may relevant to its tolerance to high salt environment. With this halotolerance property, PE10 could be a candidate for industrial use.

Lens culinaris: A new biocatalyst for reducing carbonyl and nitro groups

A series of aliphatic and aromatic aldehydes and ketones, as well as some nitrocompounds were reduced using whole plant cells from Lens culinaris seeds. In addition, we also investigated the possibility of enzymatic ester hydrolysis to explore the potential of these seeds. The reduced ketones products were obtained in yields of 8 ∼ 82% and enantiomeric excess of 39 ∼ 75%. Aldehydes were more reactive than ketones with high chemical yield (95→99%), whereas the aromatic nitrocompounds showed low (2%) to high (> 99%) conversion depending upon the nature and position of the aromatic ring substituents. Ester hydrolysis by the Lens culinaris was quite effective with the ester p-nitrophenyl acetate (> 99% conversion).

Sugar Ester Synthesis by Thermostable Lipase from Streptomyces thermocarboxydus ME168

The extracellular lipase from Streptomyces thermocarboxydus ME168 was purified to 9.5-fold with 20% yield, following concentration by acetone precipitation, ion exchange chromatography (Resource Q) and gel filtration chromatography (Superdex 200), respectively. The purified enzyme had an apparent molecular mass of 21 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The N-terminal sequence of the lipase was ASDFDDQILG and was different from most other reported lipase. The enzyme showed maximum activity at 50 °C with the half-life of 180 min at 65 °C. It showed high stability at a broad pH range of 5.5-9.5 and was thermostable at the temperature range of 25-60 °C. The K(m) and V(max) were 0.28 mM and 1,428 U/mg, respectively, using p-nitrophenyl palmitate as substrate. It was active toward p-nitrophenyl ester with medium to long acyl chain (C(8)-C(16)). Lipase activity was inhibited by Zn(2+), dithiothreitol (DTT), EDTA and some organic solvents, e.g., ethanol, acetone, dioxane, acetronitrile, tert-butanol and pyridine. Immobilized crude lipase of S. thermocarboxydus ME168 on celite could be used to synthesize sugar esters from glucose and vinyl acetate, vinyl butyrate or vinyl caproate in tert-butanol:pyridine (55:45 v/v) at 45 °C with conversion yields of 93, 67 and 55%, respectively.

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.

Purification and characterization of a new alkali-thermostable lipase from Staphylococcus aureus isolated from Arachis hypogaea rhizosphere

An extracellular lipase (EC 3.1.1.3), SAL-PP1, from Staphylococcus aureus isolated from Arachis hypogaea rhizosphere was purified and characterized. The enzyme was purified using PALL'S Microsep centrifugal device (10kD cut off), hydrophobic interaction (phenyl sepharose CL-4B column) and Superose-12 gel filtration chromatography and found to have a molecular mass of around 49kDa. The gene fragment encoding the part of the catalytic site of the SAL-PP1 lipase was sequenced and the deduced amino acid sequence shows 93% identity with that of SEL3. SAL-PP1 showed activity against long acyl-chain triglycerides, various p-nitrophenyl esters and phospholipids. The enzyme shows high stability and activity after incubation with various metal ions (retained >90% activity in presence of Ca2+, Na+, Cu2+, Mg2+, Fe2+, or Hg2+ at 10mM), organic solvents (retained >80% activity in presence of acetonitrile, ethanol, DMSO, methanol, isopropanol, toluene, or ethylene glycol at 10mM), detergents (retained >70% activity in Triton X-100, Tween 80, or sodium deoxycholate at 10mM) and irreversible inhibitors (retained >77% activity in presence of PMSF, leupetin, or β-mercaptoethanol, at 1mM). Thermal inactivation studies revealed a temperature dependent unfolding of secondary structure of protein. SAL-PP1 showed maximal activity and stability at pH 8.0 and pH 9.0, respectively. The alkali-thermostability, organic solvent-tolerance and broad substrate specificity of this enzyme may have potential implications in detergent formulations, biotransformation, industries, and medicine.