P-nitrophenyl Acetate Research Articles

Enzyme-functionalized magnetic framework composite fabricated by one-pot encapsulation of lipase and Fe3O4 nanoparticle into metal–organic framework

Introducing magnetic nanoparticles (MNP) into the enzyme@MOF complex is one of the most promising ways to facilitate enzyme recovery but typically hampered by its complex synthetic process and low loading rate. Using lipase as an example, herein, we reported a mild and quick one-pot strategy to achieve the successful encapsulation of lipase (CRL) and Fe3O4 nanoparticle into ZIF-8 to form CRL/MNP@ZIF-8 with 88.4 % immobilization yield. It is the first time that magnetic nanoparticles and lipase were simultaneously encapsulated in ZIF‐8 through the co‐precipitation approach. The resulting magnetic MOF composite demonstrated an excellent protective capability against harsh environments and could also simplify the complex downstream separation and recovery operations. Moreover, after five successive cycles of reuse to hydrolyze p-nitrophenyl acetate, the residual CRL/MNP@ZIF-8 still reserve 82 % activity, which can significantly reduce the processing cost and might be applicable in the industries in the future.

Testing for Lipase Activity in Enzymes of Unknown Function

There are over 3800 structures in the Protein Data Bank (PDB) that have unknown function. The Biochemistry Authentic Scientific Inquiry Lab (BASIL) curriculum uses authentic inquiry to teach students to use structural bioinformatics tools to compare these structures to known enzymes and predict a function. We have predicted lipase function for several structures after performing both global and local structure alignments using Dali and ProMOL. This function was supported by sequence alignment data from BLAST and PFam. Autodock Vina and PyRx were then used for ligand docking to determine possible lipid substrates to test activity. Students next used standard wet-lab biochemistry techniques to express and purify the target enzymes and performed kinetic assays first using p-nitrophenyl acetate to test for esterase activity. After completing the BASIL curriculum, students continued with independent research projects to develop assays to test for lipase function. We have focused our efforts on the protein structures with PDB IDs 2O14 and 4Q7Q, as they showed the most promise through initial enzymatic testing. Both proteins show significant esterase activity with the p-nitrophenyl acetate substrate. Additionally, both proteins show significant hydrolysis of p-nitrophenyl dodecanoate when sodium deoxycholate is present in the buffer solution for solubility of the substrate. We have also incorporated the p-nitrophenyl dodecanoate in DMPC vesicles, and both proteins can hydrolyze the substrate from the vesicle. We have tested pancreatic lipase under all these conditions as a positive control for the experiments. Students will eventually be able to update the PDB with the demonstrated function for these proteins, and they will also create a Proteopedia page for each protein. This project is supported in part by NSF IUSE 1709355.

Effect of medium on reactivity for alkaline hydrolysis of p-nitrophenyl acetate and S-p-nitrophenyl thioacetate in DMSO–H2O mixtures of varying compositions: ground state and transition state contributions

Second-order rate constants (kN) for reactions of p-nitrophenyl acetate (1) and S-p-nitrophenyl thioacetate (2) with OH– have been measured spectrophotometrically in DMSO–H2O mixtures of varying compositions at 25.0 ± 0.1 °C. The kN value increases from 11.6 to 32 800 M–1 s–1 for the reactions of 1 and from 5.90 to 190 000 M–1 s–1 for those of 2 as the reaction medium changes from H2O to 80 mol % DMSO, indicating that the effect of medium on reactivity is more remarkable for the reactions of 2 than for those of 1. Although 2 possesses a better leaving group than 1, the former is less reactive than the latter by a factor of 2 in H2O. This implies that expulsion of the leaving group is not advanced in the rate-determining transition state, i.e., the reactions of 1 and 2 with OH– proceed through a stepwise mechanism, in which expulsion of the leaving group from the addition intermediate occurs after the rate-determining step. Addition of DMSO to H2O would destabilize OH– through electronic repulsion between the anion and the negative-dipole end in DMSO. However, destabilization of OH– in the ground state is not solely responsible for the remarkably enhanced reactivity upon addition of DMSO to the medium. The effect of medium on reactivity has been dissected into the ground state and transition state contributions through combination of the kinetic data with the transfer enthalpies (ΔΔHtr) from H2O to DMSO–H2O mixtures for OH– ion.

Role of Human Arylacetamide Deacetylase (AADAC) on Hydrolysis of Eslicarbazepine Acetate and Effects of AADAC Genetic Polymorphisms on Hydrolase Activity.

Human arylacetamide deacetylase (AADAC) plays a role in the detoxification or activation of drugs and is sometimes involved in the incidence of toxicity by catalyzing hydrolysis reactions. AADAC prefers compounds with relatively small acyl groups, such as acetyl groups. Eslicarbazepine acetate, an antiepileptic drug, is a prodrug rapidly hydrolyzed to eslicarbazepine. We sought to clarify whether AADAC might be responsible for the hydrolysis of eslicarbazepine acetate. Eslicarbazepine acetate was efficiently hydrolyzed by human intestinal and liver microsomes and recombinant human AADAC. The hydrolase activities in human intestinal and liver microsomes were inhibited by epigallocatechin gallate, a specific inhibitor of AADAC, by 82% and 88% of the control, respectively. The hydrolase activities in liver microsomes from 25 human livers were significantly correlated (r = 0.87, P < 0.001) with AADAC protein levels, suggesting that the enzyme AADAC is responsible for the hydrolysis of eslicarbazepine acetate. The effects of genetic polymorphisms of AADAC on eslicarbazepine acetate hydrolysis were examined by using the constructed recombinant AADAC variants with T74A, V172I, R248S, V281I, N366K, or X400Q. AADAC variants with R248S or X400Q showed lower activity than wild type (5% or 21%, respectively), whereas those with V172I showed higher activity than wild type (174%). Similar tendencies were observed in the other four substrates of AADAC; that is, p-nitrophenyl acetate, ketoconazole, phenacetin, and rifampicin. Collectively, we found that eslicarbazepine acetate is specifically and efficiently hydrolyzed by human AADAC, and several AADAC polymorphic alleles would be a factor affecting the enzyme activity and drug response. SIGNIFICANCE STATEMENT: This is the first study to clarify that arylacetamide deacetylase (AADAC) is responsible for the activation of eslicarbazepine acetate, an antiepileptic prodrug, to eslicarbazepine, an active form, in the human liver and intestines. In addition, we found that several AADAC polymorphic alleles would be a factor affecting the enzyme activity and drug response.

Modified silicates and carbon nanotubes for immobilization of lipase from Rhizomucor miehei: Effect of support and immobilization technique on the catalytic performance of the immobilized biocatalysts

Lipase from Rhizomucor miehei (RML) was covalently immobilized on different supports, two silica gels and two carbon nanotube samples, using two different strategies. RML was immobilized on 3-carboxypropyl silica gel (RML@Si−COOH) and multi-wall carbon nanotubes containing carboxylic acid functionalities (RML@MCNT−COOH) using a two-step carbodiimide activation/immobilization reaction. Moreover, the enzyme was also immobilized on 3-aminopropyl silica (RML@Si-Glu) and single-wall carbon nanotubes functionalized with 3-APTES and activated with glutaraldehyde (RML@SCNT-Glu). Before and after RML immobilization, the structurel properties of supports were characterized and compared in detail. After immobilization, the expressed activities were 36.9, 90.2, 16.9, and 26.1 % for RML@Si−COOH, RML@Si-Glu, RML@MCNT−COOH, and RML@SCNT-Glu, respectively. The kinetic parameters of free and immobilized RML samples were determined for three substrates, p-nitrophenyl acetate, p-nitrophenyl butyrate and p-nitrophenyl palmitate, and RML@Si-Glu showed higher catalytic efficiency than the other immobilized RML samples. RML@Si−COOH, RML@Si-Glu, RML@MCNT−COOH, and RML@SCNT-Glu exhibited 5.8, 7.6, 4.2 and 4.6 folds longer half-life values than those of the free enzyme at pH 7.5 and 40 °C. Recyclability studies showed that all the immobilized RML biocatalysts retained over 90 % of their initial activities after ten cycles in the hydrolysis of p-nitrophenyl butyrate.

Rotating bed reactor packed with heterofunctional structured silica-supported lipase. Developing an effective system for the organic solvent and aqueous phase reactions

Abstract Production of specialty chemicals increasingly makes use of the enzyme catalysts in organic solvents, and Novozym 435 (N435) is among most often applied. However, its polymeric skeleton is unstable in many solvents. In this context we report the results of a more systematic study of the biocatalysts, fabricated using highly porous siliceous pellets/enzyme carriers (MH, 4 cm3 g−1 total pore volume, 310 m2 g−1 surface area), grafted with octyl (-O), amino (-A) and octyl and amino (-OA) groups, to attach the enzyme - CALB lipase. The catalysts were deployed in a rotating bed reactor (RBR) and tested in hydrolysis of p-nitrophenyl acetate, and esterification of levulinic acid. Performance of the system was compared with the same RBR filled with N435. N435 appeared the most active in both reactions, when activity was related to bulk mass of the catalysts, mainly owing to very large enzyme load. But its pore structure degraded in many typical solvents already after 24 h of soaking, whereas no significant change in MH-O and MH-OA activity was detected. Irrespective of the solvent and reaction, the highest specific activity of the enzyme featured MH-O. However, a significant enzyme leaching from MH-O observed in a hydrolytic reaction, in stark contrast to MH-OA which came second in specific activity, point to MH-OA as the catalyst of choice for hydrolytic reactions. In esterification reaction the MH-O lipase was not only most active but it was also quite stable. Therefore, MH-O appears the most suitable production system for the reactions carried out in organic solvents.

Influence of cationic surfactants and inorganic salts on the enzyme kinetic activity of Mucor javanicus lipase

AbstractIn this article, the enzyme kinetic activity of Mucor javanicus lipase (MJL) has investigated via hydrolysis of p‐nitrophenyl acetate (PNPA) in different pH environment, cationic surfactants, and inorganic salts. The influence of hydrophilicity has been studied in the presence of cationic cetyltrimethylammonium bromide (CTAB), cetyltriphenylphosphonium bromide (CTPB), and cetyldimethylethanolammonium bromide (CDMEAB) surfactants. The head group dimensions of surfactants show noteworthy contributions to enhance the catalytic activity of MJL, that is the CDMEAB surfactant, which is more efficient when compared with CTPB and CTAB. The increase in hydrophobicity of alkyltrimethylammonium bromide and alkyltriphenylphosphonium bromide in series dodecyl (C12) &lt; tetradecyl (C14) &lt; cetyl (C16) leads to a marked decrease in the observed enzymatic superactivity, but an opposite trend appears in case of alkyldimethylammonium bromide. The activity of MJL for the hydrolysis of PNPA was higher in the presence of sodium chloride and potassium chloride anion due to specific interactions between the lipase surface and anions of the Hofmeister effect.

A De Novo Designed Esterase with p-Nitrophenyl Acetate Hydrolysis Activity

Esterases are a large family of enzymes with wide applications in the industry. However, all esterases originated from natural sources, limiting their use in harsh environments or newly- emerged reactions. In this study, we designed a new esterase to develop a new protocol to satisfy the needs for better biocatalysts. The ideal spatial conformation of the serine catalytic triad and the oxygen anion hole at the substrate-binding site was constructed by quantum mechanical calculation. The catalytic triad and oxygen anion holes were then embedded in the protein scaffold using the new enzyme protocol in Rosetta 3. The design results were subsequently evaluated, and optimized designs were used for expression and purification. The designed esterase had significant lytic activities towards p-nitrophenyl acetate, which was confirmed by point mutations. Thus, this study developed a new protocol to obtain novel enzymes that may be useful in unforgiving environments or novel reactions.

Characterization of a novel hyper-thermostable and chlorpyrifos-hydrolyzing carboxylesterase EstC: A representative of the new esterase family XIX

Carboxylesterases have widely been used in a series of industrial applications, especially, the detoxification of pesticide residues. In the present study, EstC, a novel carboxylesterase from Streptomyces lividans TK24, was successfully heterogeneously expressed, purified and characterized. Phylogenetic analysis showed that EstC can be assigned as the first member of a novel family XIX. Multiple sequence alignment indicated that EstC has highly conserved structural features, including a catalytic triad formed by Ser155, Asp248 and His278, as well as a canonical Gly-His-Ser-Ala-Gly pentapeptide. Biochemical characterization indicated that EstC exhibited maximal activity at pH 9.0 (Tris-HCl buffer) and 55 °C. It also showed higher activity towards short-chain substrates, with the highest activity for p-nitrophenyl acetate (pNPA2) (Km = 0.31 ± 0.02 mM, kcat/Km = 1923.35 ± 9.62 s−1 mM−1) compared to other pNP esters used in this experiment. Notably, EstC showed hyper-thermostability and good alkali stability. The activity of EstC had no significant changes when it was incubated under 55 °C for 100 h and reached half-life after incubation at 100 °C for 8 h. Beyond that, EstC also showed stability at pH ranging from 6.0 to 11.0 and about 90% residual activity still reserved after treatment at pH 8.0 or 9.0 for 26 h, especially. Furthermore, EstC had outstanding potential for bioremediation of chlorpyrifos-contaminated environment. The recombinant enzyme (0.5 U mL−1) could hydrolyze 79.89% chlorpyrifos (5 mg L−1) at 37 °C within 80 min. These properties will make EstC have a potential application value in various industrial productions and detoxification of chlorpyrifos residues.

Making or Breaking Metal-Dependent Catalytic Activity: The Role of Stammers in Designed Three-Stranded Coiled Coils.

While many life-critical reactions would be infeasibly slow without metal cofactors, a detailed understanding of how protein structure can influence catalytic activity remains elusive. Using de novo designed three-stranded coiled coils (TRI and Grand peptides formed using a heptad repeat approach), we examine how the insertion of a three residue discontinuity, known as a stammer insert, directly adjacent to a (His)3 metal binding site alters catalytic activity. The stammer, which locally alters the twist of the helix, significantly increases copper-catalyzed nitrite reductase activity (CuNiR). In contrast, the well-established zinc-catalyzed carbonic anhydrase activity (p-nitrophenyl acetate, pNPA) is effectively ablated. This study illustrates how the perturbation of the protein sequence using non-coordinating and non-acid base residues in the helical core can perturb metalloenzyme activity through the simple expedient of modifying the helical pitch adjacent to the catalytic center.

Resistance to commonly used insecticides and underlying mechanisms of resistance in Aedes aegypti (L.) from Sri Lanka

BackgroundDrastic increases of dengue fever (DF) over the past few years have prompted studies on the development of resistance to insecticides in the mosquito vector, Aedes aegypti (Linnaeus). In Sri Lanka control of the vector population is essentially achieved using larvicides (temephos) and adulticides (principally pyrethroids). The present study investigates resistance to commonly used insecticides and underlying mechanisms of Ae. aegypti in selected sites in Sri Lanka.MethodsIn this study, susceptibility to three commonly used adulticides (malathion, permethrin and deltamethrin) and the larvicide temephos were tested for Ae. aegypti sampled from five localities in Sri Lanka using WHO dose diagnostics tests. In addition, we performed dose-response tests for permethrin to determine lethal concentrations (LCs) with CDC bottle bioassays. An assessment of the activity of metabolic detoxifying enzymes (multifunction oxidases (MFOs), glutathione S-transferases (GSTs) and esterases) and determination of frequency of the kdr mutations (F1534C, V1016G and S989P) were also carried out to ascertain the associated resistance mechanisms. Kdr genotype frequencies were compared with samples collected from the same sites in 2015 to determine the change of allele frequencies over the years.ResultsThe present study revealed resistance in all Ae. aegypti populations studied, with low mortality percentages for both permethrin (10–89%) and deltamethrin (40–92%). Dose response tests revealed highest resistance ratios (RR) for permethrin and temephos from Colombo district whereas Puttalum district exhibited the lowest. High frequencies of the 1534C allele (0.052–0.802) were found in the study sites in 2017. Comparison with samples collected in 2015 revealed a substantial increase in this allele. The activity of MFOs and p-nitro phenyl-acetate esterase was significantly greater in most Sri Lankan populations in comparison to that of the New Orleans (NO) susceptible strain. In contrast, the activity of α-esterase and β-esterase was similar or lower than that in the NO strain.ConclusionsAedes aegypti from Sri Lanka is resistant to pyrethroid insecticides showing rapid selection for kdr mutations and varying metabolic mechanisms. Continued monitoring of vector populations is crucial to mitigate the development of resistance to commonly used insecticides and in turn, controlling the vector population.

Characterization of XtjR8: A novel esterase with phthalate-hydrolyzing activity from a metagenomic library of lotus pond sludge

A fosmid metagenomic library containing 9.7 × 104 clones was constructed. A novel esterase, XtjR8, was isolated through functional screening. XtjR8 shared the maximum amino acid identity (44%) with acetyl-hydrolase from Streptomyces hygroscopicus, and was classified into family IV esterase. XtjR8 exhibited the highest hydrolytic activity for p-nitrophenyl acetate at 40 °C and pH 8.0, and presented more than 40% activity from 20 °C to 80 °C. More importantly, XtjR8 displayed the ability to hydrolyze both phthalate monoesters and diesters, this feature is extremely rare among previously reported esterases. Site-directed mutagenesis experiments revealed that the catalytic triad residues were Ser152, Glu246, and His276. Among them, Ser152 formed a hydrogen bond with dibutyl phthalate (DBP) by molecular docking, Gly84, Gly85, and Leu248 of conserved motifs formed hydrophobic interactions with DBP, respectively, which were important for the catalytic activity. Considering its wide range of temperature and hydrolytic potential toward phthalate esters, XtjR8 will be served as an interesting candidate for biodegradation and industrial applications.

Larger active site in an ancestral hydroxynitrile lyase increases catalytically promiscuous esterase activity.

Hydroxynitrile lyases (HNL's) belonging to the α/β-hydrolase-fold superfamily evolved from esterases approximately 100 million years ago. Reconstruction of an ancestral hydroxynitrile lyase in the α/β-hydrolase fold superfamily yielded a catalytically active hydroxynitrile lyase, HNL1. Several properties of HNL1 differ from the modern HNL from rubber tree (HbHNL). HNL1 favors larger substrates as compared to HbHNL, is two-fold more catalytically promiscuous for ester hydrolysis (p-nitrophenyl acetate) as compared to mandelonitrile cleavage, and resists irreversible heat inactivation to 35 °C higher than for HbHNL. We hypothesized that the x-ray crystal structure of HNL1 may reveal the molecular basis for the differences in these properties. The x-ray crystal structure solved to 1.96-Å resolution shows the expected α/β-hydrolase fold, but a 60% larger active site as compared to HbHNL. This larger active site echoes its evolution from esterases since related esterase SABP2 from tobacco also has a 38% larger active site than HbHNL. The larger active site in HNL1 likely accounts for its ability to accept larger hydroxynitrile substrates. Site-directed mutagenesis of HbHNL to expand the active site increased its promiscuous esterase activity 50-fold, consistent with the larger active site in HNL1 being the primary cause of its promiscuous esterase activity. Urea-induced unfolding of HNL1 indicates that it unfolds less completely than HbHNL (m-value = 0.63 for HNL1 vs 0.93 kcal/mol·M for HbHNL), which may account for the ability of HNL1 to better resist irreversible inactivation upon heating. The structure of HNL1 shows changes in hydrogen bond networks that may stabilize regions of the folded structure.

Biocatalysis of D,L-Peptide Nanofibrillar Hydrogel.

Self-assembling peptides are attracting wide interest as biodegradable building blocks to achieve functional nanomaterials that do not persist in the environment. Amongst the many applications, biocatalysis is gaining momentum, although a clear structure-to-activity relationship is still lacking. This work applied emerging design rules to the heterochiral octapeptide sequence His–Leu–DLeu–Ile–His–Leu–DLeu–Ile for self-assembly into nanofibrils that, at higher concentration, give rise to a supramolecular hydrogel for the mimicry of esterase-like activity. The peptide was synthesized by solid-phase and purified by HPLC, while its identity was confirmed by 1H-NMR and electrospray ionization (ESI)-MS. The hydrogel formed by this peptide was studied with oscillatory rheometry, and the supramolecular behavior of the peptide was investigated with transmission electron microscopy (TEM) analysis, circular dichroism (CD) spectroscopy, thioflavin T amyloid fluorescence assay, and attenuated total reflectance (ATR) Fourier-transform infrared (FT-IR) spectroscopy. The biocatalytic activity was studied by monitoring the hydrolysis of p-nitrophenyl acetate (pNPA) at neutral pH, and the reaction kinetics followed an apparent Michaelis–Menten model, for which a Lineweaver–Burk plot was produced to determine its enzymatic parameters for a comparison with the literature. Finally, LC–MS analysis was conducted on a series of experiments to evaluate the extent of, if any, undesired peptide acetylation at the N-terminus. In conclusion, we provide new insights that allow gaining a clearer picture of self-assembling peptide design rules for biocatalysis.

Zn-triazole coordination polymers: Bioinspired carbonic anhydrase mimics for hydration and sequestration of CO2

Artificial enzyme mimics have recently emerged as alternative biocatalysts for overcoming the intrinsic fragility of natural enzyme in practical applications. However, current researches regarding mimetic enzymes are still confined to very few reaction types, with artificial oxidoreductases as dominance. Herein, inspired by nature, we designed and fabricated a range of Zn-Triazole coordination polymers (ZnTazs) that presented similar coordination structures with the active site of natural carbonic anhydrase Ⅱ (CA Ⅱ). These synthesized compounds exhibited inherently mimetic function with natural CA for catalyzing the hydrolysis of p-nitrophenyl acetate (p-NPA). Especially, the initial hydrolysis rate (V0) of p-NPA catalyzed by ZnTaz-1 (Zn5(bta)6(NO3)4 · H2O) and ZnTaz-2 (Zn3(OH)2(btca)2 · DMF · 4 H2O) reached 42.1 and 73.8 nM · s−1, respectively. Meanwhile, ZnTaz-1 and ZnTaz-2 showed favorable recyclability, and excellent stability towards various pH values and organic solvents, which are of great significance for practical employment. Moreover, they could also promote the efficient hydration and sequestration of greenhouse gas CO2 in aqueous medium. Based on this work, we aim to provide more theoretical and practical basis for rational design of CA mimics from the inspiration of natural enzyme, as well as propose a potential strategy for tackling the CO2 crisis.

Macroporous Silica Microcapsules Immobilizing Esterase with High Hydrolysis Reactivity

Abstract An esterase, 3,4-dihydrocoumarin hydrolase, was directly immobilized into silica microcapsules. The hydrolysis reaction of 3,4-dihydrocoumarin by a macroporous silica microcapsule immobilizing the esterase was faster than those by mesoporous ones. Using this macroporous microcapsule, the hydrolysis reaction of p-nitrophenyl acetate proceeded with comparable rate to non-immobilized esterase.

Smart bilayer polymer reactor with cascade/non-cascade switching catalyst characteristics

In this work, a new method is proposed to meet the challenge of preparing new catalysts with cascade/non-cascade switching catalytic property. Inspired from “soft” characteristics and divisional isolation function in natural biological systems, this objective was accomplished by developing a new class of hydrogels made of two unique functional layers with different temperature responses where each may self-govern coupled processes at a specific temperature. This hydrogel polymer reactor exhibited almost no catalytic activity at low-temperature range (<37 °C) as both channels of bilayer hydrogel polymer catalyst were closed. At modest temperatures (between 37 °C and 50 °C), the first step of the tandem reaction (the hydrolysis of p-nitrophenyl acetate (NPA)) showed significant reactivity that arises from the relaxing of the weak polymer complexes in the hydrogel layer. This enabled NPA the access to the acidic catalytic active center of the hydrogel. At range of higher temperatures (>50 °C), the hydrogel catalytic polymer reactor further exhibited significant efficiency towards the hydrolysis reaction of NPA as well as the reduction of the intermediate product p-nitrophenol (NP). This mainly resulted from the opening of both the weak polymer complexes and the stronger polymer complexes hydrogel layers, allowing entrance to both the acidic catalytic active center and the metal nanoparticles active center. As a result, the novel hydrogel polymer reactor could be used to control cascade/non-cascade catalysis reactions. This new protocol enables efficient control of switchable tandem reactions, inspiring for difficulty to control tandem catalytic reactors.

Effect of CTAB reverse micelles on the kinetics of aminolysis of p-nitrophenyl acetate by hydrazine

Effect of CTAB reverse micelles on the kinetics of aminolysis of p-nitrophenyl acetate by hydrazine

Functional Expression and Characterization of Acetyl Xylan Esterases CE Family 7 from Lactobacillus antri and Bacillus halodurans.

Acetyl xylan esterase (AXE; E.C. 3.1.1.72) is one of the accessory enzymes for xylan degradation, which can remove the terminal acetate residues from xylan polymers. In this study, two genes encoding putative AXEs (LaAXE and BhAXE) were cloned from Lactobacillus antri DSM 16041 and Bacillus halodurans C-125, and constitutively expressed in Escherichia coli. They possess considerable activities towards various substrates such as p-nitrophenyl acetate, 4-methylumbelliferyl acetate, glucose pentaacetate, and 7-amino cephalosporanic acid. LaAXE and BhAXE showed the highest activities at pH 7.0 and 8.0 at 50°C, respectively. These enzymes are AXE members of carbohydrate esterase (CE) family 7 with the cephalosporine-C deacetylase activity for the production of antibiotics precursors. The simultaneous treatment of LaAXE with Thermotoga neapolitana β-xylanase showed 1.44-fold higher synergistic degradation of beechwood xylan than the single treatment of xylanase, whereas BhAXE showed no significant synergism. It was suggested that LaAXE can deacetylate beechwood xylan and enhance the successive accessibility of xylanase towards the resulting substrates. The novel LaAXE originated from a lactic acid bacterium will be utilized for the enzymatic production of D-xylose and xylooligosaccharides.

Characterization of Novel Salt-Tolerant Esterase Isolated from the Marine Bacterium Alteromonas sp. 39-G1.

An esterase gene, estA1, was cloned from Alteromonas sp. 39-G1 isolated from the Beaufort Sea. The gene is composed of 1,140 nucleotides and codes for a 41,190 Da protein containing 379 amino acids. As a result of a BLAST search, the protein sequence of esterase EstA1 was found to be identical to Alteromonas sp. esterase (GenBank: PHS53692). As far as we know, no research on this enzyme has yet been conducted. Phylogenetic analysis showed that esterase EstA1 was a member of the bacterial lipolytic enzyme family IV (hormone sensitive lipases). Two deletion mutants (Δ20 and Δ54) of the esterase EstA1 were produced in Escherichia coli BL21 (DE3) cells with part of the N-terminal of the protein removed and His-tag attached to the C-terminal. These enzymes exhibited the highest activity toward p-nitrophenyl (pNP) acetate (C2) and had little or no activity towards pNP-esters with acyl chains longer than C6. Their optimum temperature and pH of the catalytic activity were 45°C and pH 8.0, respectively. As the NaCl concentration increased, their enzyme activities continued to increase and the highest enzyme activities were measured in 5 M NaCl. These enzymes were found to be stable for up to 8 h in the concentration of 3-5 M NaCl. Moreover, they have been found to be stable for various metal ions, detergents and organic solvents. These salt-tolerant and chemical-resistant properties suggest that the enzyme esterase EstA1 is both academically and industrially useful.