Ester Substrates Research Articles

Sulfur K-edge X-ray absorption spectroscopy and density functional theory calculations on monooxo Mo(IV) and bisoxo Mo(VI) bis-dithiolenes: insights into the mechanism of oxo transfer in sulfite oxidase and its relation to the mechanism of DMSO reductase.

Sulfur K-edge X-ray absorption spectroscopy (XAS) and density functional theory (DFT) calculations have been used to determine the electronic structures of two complexes [MoIVO(bdt)2]2– and [MoVIO2(bdt)2]2– (bdt = benzene-1,2-dithiolate(2−)) that relate to the reduced and oxidized forms of sulfite oxidase (SO). These are compared with those of previously studied dimethyl sulfoxide reductase (DMSOr) models. DFT calculations supported by the data are extended to evaluate the reaction coordinate for oxo transfer to a phosphite ester substrate. Three possible transition states are found with the one at lowest energy, stabilized by a P–S interaction, in good agreement with experimental kinetics data. Comparison of both oxo transfer reactions shows that in DMSOr, where the oxo is transferred from the substrate to the metal ion, the oxo transfer induces electron transfer, while in SO, where the oxo transfer is from the metal site to the substrate, the electron transfer initiates oxo transfer. This difference in reactivity is related to the difference in frontier molecular orbitals (FMO) of the metal–oxo and substrate–oxo bonds. Finally, these experimentally related calculations are extended to oxo transfer by sulfite oxidase. The presence of only one dithiolene at the enzyme active site selectively activates the equatorial oxo for transfer, and allows facile structural reorganization during turnover.

Mechanistic study of chemoselectivity in Ni-catalyzed coupling reactions between azoles and aryl carboxylates.

Itami et al. recently reported the C-O electrophile-controlled chemoselectivity of Ni-catalyzed coupling reactions between azoles and esters: the decarbonylative C-H coupling product was generated with the aryl ester substrates, while C-H/C-O coupling product was generated with the phenol derivative substrates (such as phenyl pivalate). With the aid of DFT calculations (M06L/6-311+G(2d,p)-SDD//B3LYP/6-31G(d)-LANL2DZ), the present study systematically investigated the mechanism of the aforementioned chemoselective reactions. The decarbonylative C-H coupling mechanism involves oxidative addition of C(acyl)-O bond, base-promoted C-H activation of azole, CO migration, and reductive elimination steps (C-H/Decar mechanism). This mechanism is partially different from Itami's previous proposal (Decar/C-H mechanism) because the C-H activation step is unlikely to occur after the CO migration step. Meanwhile, C-H/C-O coupling reaction proceeds through oxidative addition of C(phenyl)-O bond, base-promoted C-H activation, and reductive elimination steps. It was found that the C-O electrophile significantly influences the overall energy demand of the decarbonylative C-H coupling mechanism, because the rate-determining step (i.e., CO migration) is sensitive to the steric effect of the acyl substituent. In contrast, in the C-H/C-O coupling mechanism, the release of the carboxylates occurs before the rate-determining step (i.e., base-promoted C-H activation), and thus the overall energy demand is almost independent of the acyl substituent. Accordingly, the decarbonylative C-H coupling product is favored for less-bulky group substituted C-O electrophiles (such as aryl ester), while C-H/C-O coupling product is predominant for bulky group substituted C-O electrophiles (such as phenyl pivalate).

Structural characterization and function determination of a nonspecific carboxylate esterase from the amidohydrolase superfamily with a promiscuous ability to hydrolyze methylphosphonate esters.

The uncharacterized protein Rsp3690 from Rhodobacter sphaeroides is a member of the amidohydrolase superfamily of enzymes. In this investigation the gene for Rsp3690 was expressed in Escherichia coli and purified to homogeneity, and the three-dimensional structure was determined to a resolution of 1.8 Å. The protein folds as a distorted (β/α)8-barrel, and the subunits associate as a homotetramer. The active site is localized to the C-terminal end of the β-barrel and is highlighted by the formation of a binuclear metal center with two manganese ions that are bridged by Glu-175 and hydroxide. The remaining ligands to the metal center include His-32, His-34, His-207, His-236, and Asp-302. Rsp3690 was shown to catalyze the hydrolysis of a wide variety of carboxylate esters, in addition to organophosphate and organophosphonate esters. The best carboxylate ester substrates identified for Rsp3690 included 2-naphthyl acetate (kcat/Km = 1.0 × 105 M–1 s–1), 2-naphthyl propionate (kcat/Km = 1.5 × 105 M–1 s–1), 1-naphthyl acetate (kcat/Km = 7.5 × 103 M–1 s–1), 4-methylumbelliferyl acetate (kcat/Km = 2.7 × 103 M–1 s–1), 4-nitrophenyl acetate (kcat/Km = 2.3 × 105 M–1 s–1), and 4-nitrophenyl butyrate (kcat/Km = 8.8 × 105 M–1 s–1). The best organophosphonate ester substrates included ethyl 4-nitrophenyl methylphosphonate (kcat/Km = 3.8 × 105 M–1 s–1) and isobutyl 4-nitrophenyl methylphosphonate (kcat/Km = 1.1 × 104 M–1 s–1). The (SP)-enantiomer of isobutyl 4-nitrophenyl methylphosphonate was hydrolyzed 10 times faster than the less toxic (RP)-enantiomer. The high inherent catalytic activity of Rsp3690 for the hydrolysis of the toxic enantiomer of methylphosphonate esters make this enzyme an attractive target for directed evolution investigations.

Caffeic acid O-methyltransferase from Leucaena leucocephala: Cloning, expression, characterization and molecular docking analyses

Abstract Angiospermic caffeic acid O-methyltransferase (COMT) conventionally catalyzes the methylation of caffeic acid as well as 5-hydroxy ferulic acid to ferulic acid and sinapic acid, respectively, using S-adenosyl- l -methionine (SAM) as methyl group donor. A cDNA of OMT from Leucaena leucocephala (LlOMT) was cloned, expressed and purified. The expressed protein was purified to homogeneity on Ni–NTA agarose column with specific activity of 450 nmol of ferulic acid formed/min/mg protein. Native molecular weight of the purified enzyme was found to be 80 kDa and that of subunit molecular weight was found to be 40 kDa, suggesting the homodimeric nature of the enzyme. The optimum temperature and pH was found to be 37 °C and pH 8.0, respectively. Apparent K m of enzyme for caffeic acid and SAM was found to be 220 μM and 2.12 μM, respectively. In view of overlapping/metabolic grid concept of methylation of substrates in phenyl propanoid pathway, an in silico approach was used to look into it. Accordingly, the LlOMT protein was modeled and docked with 16 putative substrates (intermediates of phenyl propanoid/monolignol biosynthesis pathway). In silico analyses revealed that the Co-A ester substrates were most favored among those of substrates, analyzed.

Agreement of serum feline pancreas-specific lipase and colorimetric lipase assays with pancreatic ultrasonographic findings in cats with suspicion of pancreatitis: 161 cases (2008-2012).

To investigate agreement of a feline pancreas-specific lipase assay and a colorimetric lipase assay with a 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester (DGGR) substrate with results of pancreatic ultrasonography in cats with suspicion of pancreatitis. Retrospective case series. 161 client-owned cats with suspicion of pancreatitis. Feline pancreas-specific lipase concentration and DGGR lipase activity were measured from the same blood sample in cats undergoing investigation for pancreatitis, with < 24 hours between ultrasonography and lipase determinations. Ultrasonographic variables evaluated were ultrasonographic diagnosis of pancreatitis, enlargement, margins, echogenicity, mesenteric echogenicity, peripancreatic free fluid, cysts, masses, and common bile and pancreatic duct dilation. Agreement was assessed by use of the Cohen κ coefficient. Agreement between the lipase assays was substantial (κ = 0.703). An ultrasonographic diagnosis of pancreatitis had fair agreement with feline pancreas-specific lipase concentration > 5.4 μg/L (κ = 0.264) and DGGR lipase activity > 26 U/L (κ = 0.221). The greatest agreement between feline pancreas-specific lipase concentration > 5.4 μg/L and DGGR lipase activity > 26 U/L was found for a hypoechoic and mixed-echoic (κ = 0.270 and 0.266, respectively), hypoechoic (κ = 0.261 and 0.181, respectively), and enlarged (κ = 0.218 and 0.223, respectively) pancreas. Agreement between pancreatic ultrasonography and lipase assay results was only fair. It remains unknown whether lipase results or pancreatic ultrasonography constitutes the more accurate test for diagnosing pancreatitis; therefore, results of both tests need to be interpreted with caution.

Probing the active site of the Rv0045c esterase from Mycobaterium tuberculosis (580.4)

The active site of the Rv0045c esterase from Mycobaterium tuberculosis was probed with a combination of site‐directed mutagenesis and a series of substrate analogues. The thermal stability of enzyme variants was measured using differential scanning fluorimetry with Sypro Orange dye. The catalytic efficiency of the enzyme variants was measured using fluorogenic ester substrates. Variants of active site residues confirmed a classic serine catalytic triad, as well as revealing the relative importance of several other active site residues. Two active site variants (G90A and H187A) displayed increased catalytic efficiency relative to the wild type, but the corresponding double variant lacked a synergistic activation effect. A series of other H187 variants revealed that further fine‐tuning of enzymatic activity was possible. Additionally, the relative catalytic efficiencies of the hydrolysis of a series of flourogenic ester substrates revealed structure activity relationships, in which short chain, unbranched, hydrogen bond accepting esters displayed maximal activity.Grant Funding Source: Supported by the National Science Foundation

Two-step process for preparation of oligosaccharide propionates and acrylates using lipase and Cyclodextrin Glycosyl Transferase (CGTase)

Abstract Background Oligosaccharide esters are attractive candidates for applications as surfactants, hydrogels and other materials, but direct enzymatic acylation is difficult with carbohydrates longer than disaccharides. Results A combination of one lipase-catalyzed step and one transglycosylation step catalyzed by a cyclodextrin glycosyl transferase (CGTase) was used to synthesize oligosaccharide esters. The conversion of glucose and maltose with vinyl propionate catalyzed by Candida antarctica lipase B (Novozym 435) in dioxane proceeded to full conversion to mixtures of mono and diesters. When ethyl acrylate was used as acyl donor, mono and diesters were formed, but full conversion was not reached. The CGTase catalyzed reactions between the glucose and maltose esters and α-cyclodextrin were carried out in water. In the initial phase, addition of the glucose residues of the cyclodextrin to the ester substrate occurred (coupling reaction), followed by disproportionation reactions yielding a range of oligosaccharide esters with varying chain length. The monoesters were efficient acceptors in the CGTase-catalyzed reactions, while the diesters were not converted to a significant extent. As a consequence, the glucose propionate which contained large amounts of diesters was converted to 40% conversion while the maltose propionate which contained mainly monoesters was converted to 86% conversion. Conclusions A two-step enzymatic process for preparation of oligosaccharide esters has been developed. Oligosaccharide propionates were produced in high yield with a total reaction time of 5 h. The double bond of the acrylate moiety reduced the reaction rate of the lipase catalyzed transesterification, but in both cases, the CGTase efficiently converted the monoesters to oligosaccharide esters.

Deletion of acetyl-CoA synthetases I and II increases production of 3-hydroxypropionate by the metabolically-engineered hyperthermophile Pyrococcus furiosus

The heterotrophic, hyperthermophilic archaeon Pyrococcus furiosus is a new addition to the growing list of genetically-tractable microorganisms suitable for metabolic engineering to produce liquid fuels and industrial chemicals. P. furiosus was recently engineered to generate 3-hydroxypropionate (3-HP) from CO2 and acetyl-CoA by the heterologous-expression of three enzymes from the CO2 fixation cycle of the thermoacidophilic archaeon Metallosphaera sedula using a thermally-triggered induction system. The acetyl-CoA for this pathway is generated from glucose catabolism that in wild-type P. furiosus is converted to acetate with concurrent ATP production by the heterotetrameric (α2β2) acetyl-CoA synthetase (ACS). Hence ACS in the engineered 3-HP production strain (MW56) competes with the heterologous pathway for acetyl-CoA. Herein we show that strains of MW56 lacking the α-subunit of either of the two ACSs previously characterized from P. furiosus (ACSI and ACSII) exhibit a three-fold increase in specific 3-HP production. The ΔACSIα strain displayed only a minor defect in growth on either maltose or peptides, while no growth defect on these substrates was observed with the ΔACSIIα strain. Deletion of individual and multiple ACS subunits was also shown to decrease CoA release activity for several different CoA ester substrates in addition to acetyl-CoA, information that will be extremely useful for future metabolic engineering endeavors in P. furiosus.

Epoxidation of alkenes bearing a carboxylic acid group by iron complexes of the tetradentate ligand N,N′-dimethyl-N,N′-bis(2-pyridylmethyl)-1,2-diaminoethane and its derivatives

The addition of carboxylic acids enhances the rate and selectivity of alkene oxidations catalyzed by [(bpmen)Fe(OTf)2] (bpmen=N,N′-dimethyl-N,N′-bis(2-pyridylmethyl)-1,2-diaminoethane). The syntheses and characterizations of four derivatives of this iron complex with varying substitutions on the pyridine ring and with a substituted piperazine backbone are reported. These [(L)Fe(OTf)2] complexes and [(bpmen)Fe(OTf)2] are employed as catalysts for the oxidation of alkenes bearing a carboxylic acid functional group, namely oleic acid, undecylenic acid, 5-hexenoic acid and 4-pentenoic acid, with hydrogen peroxide as the oxidant. Comparisons with the analogous ester substrates demonstrate the beneficial impact of the acid functional group on conversion and selectivity when using [(bpmen)Fe(OTf)2] as a catalyst. For the oleic and undecylenic acids, epoxide product is formed with moderate to high conversions and high selectivities. Under the conditions employed, 4-pentenoic acid is oxidized to a γ-lactone, most likely via the epoxide intermediate, and 5-hexenoic acid to a mixture of epoxide and δ-lactone. Of the iron complexes with bpmen derivatives as ligands, only the N,N′-dimethyl-N,N′-bis(5-chloropyridin-2-ylmethyl)-1,2-diaminoethane variant shows appreciable activity. The effect of solvent choice is also investigated.

An efficient method for enzymatic purification of cis-9,trans-11 isomer of conjugated linoleic acid

Abstract A method for purification of cis-9,trans-11 isomer of conjugated linoleic acid (CLA) is described. Lipase from Candida cylindracea (CCL) was highly selective towards cis-9,trans-11 isomer and was chosen to catalyze esterification of a mixture of two CLA isomers. The effect of various parameters on the esterification process of an almost equimolar mixture of cis-9,trans-11 (44.0%), trans-10,cis-12 (42.5%) and other CLA isomers (13.5%) was studied. The influence of alcohol substrate, CLA/alcohol molar ratio, lipase amount, reaction temperature, pH and time of the reaction on the total esterification and cis-9,trans-11 isomer purity in ester fraction were examined. The highest isomeric excess of cis-9,trans-11 isomer on trans-10,cis-12 CLA and good esterification degree was observed when the esterification condition were as follows: CLA/2-propanol molar ratio, 1:5; water, 200% wt of CLA; CCL, 24 μg of CCL/mg CLA; temperature 30 C; time of reaction 6 h and when mixture enriched in cis-9,trans-11 CLA was used as a substrate for esterification. A preparative scale purification combining crystallization and one step enzymatic process were made to afford purified cis-9,trans-11 (94.2%) with total recovery of 31%.

Novel stereoselective carbonyl reductase from Kluyveromyces marxianus for chiral alcohols synthesis

A novel nicotinamide adenine dinucleotide phosphate(NADPH)-dependent carbonyl reductase from Kluyveromyces marxianus(KmCR) was identified, which can convert various prochiral ketone esters and ketone substrates to their corresponding chiral alcohols. KmCR was over-expressed in E. coli BL21(DE3), purified to homogeneity, and characterized. The purified enzyme exhibits the highest activity at 40 °C and pH=6.0. Based on the gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) analysis, the monomeric protein was determined to have a molecular weight of approximate 39000. Vmax and Km of KmCR are 4.28 μmol·min−1·mg−1 and 0.41 mmol/L for ketone ester substrate ethyl 2-oxo-4-phenylbutyrate(OPBE), 3.09 μmol·min−1·mg−1 and 1.21 mmol/L for cofactor NADPH, respectively. Cofactor recycle was achieved by co-expression of KmCR and glucose dehydrogenase(GDH) in E. coli. Recombinant E. coli harboring KmCR and GDH showed moderate asymmetric reduction activity towards various α- and β-ketoesters, diaryl ketone substrates. In an aqueous/butyl acetate biphasic system, the whole-cell biocatalyst was used to prepare ethyl (R)-2-hydroxy-4-phenylbutanoate[(R)-HPBE] in an e.e. of 99.5% with a space-time yield of 433.6 g·L−1·d−1 and a yield of 80.3% at 270 g/L OPBE.

Organocatalysis approach to trifluoromethylation with fluoroform

The organic base methodology exploits an access to generate the “trifluoromethyl anion” for carbonyl, ester, acid halide, epoxide, deuterium donor, and carbon dioxide substrates to afford the trifluoromethylation products with good overall efficiency even in organocatalysis conditions. The NMR analysis of the mixture of fluoroform and P4-base shows no change thereof. However, on addition of electrophiles, the trifluoromethylation products were obtained efficiently.

Identification and Characterization of a Periplasmic Aminoacyl-phosphatidylglycerol Hydrolase Responsible for Pseudomonas aeruginosa Lipid Homeostasis*

Specific aminoacylation of the phospholipid phosphatidylglycerol (PG) with alanine (or with lysine) was shown to render various organisms less susceptible to antimicrobial agents and environmental stresses. In this study, we make use of the opportunistic pathogen Pseudomonas aeruginosa to decode ORF PA0919-dependent lipid homeostasis. Analysis of the polar lipid content of the deletion mutant ΔPA0919 indicated significantly enlarged levels of alanyl-PG. The resulting phenotype manifested an increased susceptibility to several antimicrobial compounds when compared with the wild type. A pH-dependent PA0919 promoter located within the upstream gene PA0920 was identified. Localization experiments demonstrated that the PA0919 protein is anchored to the periplasmic surface of the inner bacterial membrane. The recombinant overproduction of wild type and several site-directed mutant proteins in the periplasm of Escherichia coli facilitated a detailed in vitro analysis of the enzymatic PA0919 function. A series of artificial substrates (p-nitrophenyl esters of various amino acids/aliphatic acids) indicated enzymatic hydrolysis of the alanine, glycine, or lysine moiety of the respective ester substrates. Our final in vitro activity assay in the presence of radioactively labeled alanyl-PG then revealed hydrolysis of the aminoacyl linkage, resulting in the formation of alanine and PG. Consequently, PA0919 was termed alanyl-PG hydrolase. The elucidated enzymatic activity implies a new regulatory circuit for the appropriate tuning of cellular alanyl-PG concentrations.

ChemInform Abstract: Enantioselective Copper‐Catalyzed Intramolecular Phenolic O—H Bond Insertion: Synthesis of Chiral 2‐Carboxy Dihydrobenzofurans, Dihydrobenzopyrans, and Tetrahydrobenzooxepines.

AbstractThe cyclization of various diazo ester substrates in the presence of the optimal ligands (Sa)‐INH and (Sa)‐IOP is advantageous with regards to high yields, excellent enantioselectivity, and wide substrate scope.

A Csp3–Csp3 Bond Forming Reductive Condensation of α-Keto Esters and Enolizable Carbon Pronucleophiles

The preparation of densely functionalized unsymmetrical 1,4-dicarbonyl structural motifs by a phosphorus(III)-mediated reductive condensation of α-keto esters and enolizable carbon pronucleophiles is described. The reaction, which is initiated by Kukhtin-Ramirez addition of commercially available tris(dimethylamino)phosphine to the α-keto ester substrate, proceeds rapidly under mild conditions.

Production and activity of extracellular lipase from Luteibacter sp.

Microbial lipases are widely used in industrial applications due to their versatility, and the characterization of new lipase-producing microorganisms could provide new sources of these enzymes, with different specificities and better activities. In this context, we have improved lipase production by Luteibacter sp. by using basal medium supplemented with 2 % olive oil, a pH of 6 and a growth temperature of 37 °C. The enzyme extraction process with the addition of 0.25 % Tween 80 increased lipase activity. Implementation of these modifications increased lipase activity by approximately 430 %. The lipase activities produced in the culture supernatant (LCS) and extracted with Tween 80 (LCST80) were characterized. Both extracts hydrolyzed ρ-nitrophenyl (ρNP) esters with different acyl chain lengths, with a preference for short acyl lengths, and had optimum activity at 45 °C. The LCS was stable at acidic and alkaline pH, but LCST80 was only stable at alkaline pH. Methanol, SDS, Triton X-100, EDTA, and EGTA did not affect lipase activity, while divalent cations (Ca2+, Zn2+, Mg2+) - with the exception of Co2+— increased lipase activity. Both extracts showed transesterification activity on ρNP ester substrates, and both were able to hydrolyze different natural lipids. The characterization of lipase produced by Luteibacter sp. introduces this recently described genus as a new source of lipases with great biotechnological potential.

Substrate‐Dependent Stereospecificity of Tyl‐KR1: An Isolated Polyketide Synthase Ketoreductase Domain from Streptomyces fradiae

The stereospecificity of an enzymatic reaction depends on the way in which a substrate and its enantiomer bind to the active site. These binding modes cannot be easily predicted. We have studied the stereospecificity and stereoselectivity of the ketoreductase domain Tyl-KR1 of the tylactone polyketide synthase from Streptomyces fradiae by analysing the stereochemical outcome of the reduction of five different keto ester substrates. The absolute configuration of the Tyl-KR1 reduction products was determined by using vibrational circular dichroism (VCD) spectroscopy combined with quantum chemical calculations. The conversion of only one of the tested substrates, 2-methyl-3-oxovaleric acid N-acetylcysteamine thioester, afforded the expected anti-(2R,3R) configuration of the α-methyl-β-hydroxyl ester product, representing the stereochemistry observed for the physiological polyketide product tylactone. For all other substrates, which were modified with respect to the type of ester and/or the chain length (C4 instead of C5), the opposite configuration (anti-(2S,3S)) was obtained with significant enantio- and diastereoselectivity. Inversion of both stereocentres suggests completely different binding modes invoked by only minor modifications of the substrate structure.

Decoupled Roles for the Atypical, Bifurcated Binding Pocket of the ybfF Hydrolase

Serine hydrolases have diverse intracellular substrates, biological functions, and structural plasticity, and are thus important for biocatalyst design. Amongst serine hydrolases, the recently described ybfF enzyme family are promising novel biocatalysts with an unusual bifurcated substrate-binding cleft and the ability to recognize commercially relevant substrates. We characterized in detail the substrate selectivity of a novel ybfF enzyme from Vibrio cholerae (Vc-ybfF) by using a 21-member library of fluorogenic ester substrates. We assigned the roles of the two substrate-binding clefts in controlling the substrate selectivity and folded stability of Vc-ybfF by comprehensive substitution analysis. The overall substrate preference of Vc-ybfF was for short polar chains, but it retained significant activity with a range of cyclic and extended esters. This broad substrate specificity combined with the substitutional analysis demonstrates that the larger binding cleft controls the substrate specificity of Vc-ybfF. Key selectivity residues (Tyr116, Arg120, Tyr209) are also located at the larger binding pocket and control the substrate specificity profile. In the structure of ybfF the narrower binding cleft contains water molecules prepositioned for hydrolysis, but based on substitution this cleft showed only minimal contribution to catalysis. Instead, the residues surrounding the narrow binding cleft and at the entrance to the binding pocket contributed significantly to the folded stability of Vc-ybfF. The relative contributions of each cleft of the binding pocket to the catalytic activity and folded stability of Vc-ybfF provide a valuable map for designing future biocatalysts based on the ybfF scaffold.

Metagenomic mining of feruloyl esterases from termite enteric flora

A metagenome expression library was created from Trinervitermes trinervoides termite hindgut symbionts and subsequently screened for feruloyl esterase (FAE) activities, resulting in seven recombinant fosmids conferring feruloyl esterase phenotypes. The amino acid sequence lengths of the seven FAE encoding open reading frames (ORFs) ranged from 260 to 274 aa and encoded polypeptides of between 28.9 and 31.4 kDa. The highest sequence identity scores for the seven ORFs against the GenBank database were between 45 and 59 % to a number of carboxyl ester hydrolyses. The seven FAE primary structures contained sequence motifs that correspond well with a classical pentapeptide (G-x-S-x-G) serine hydrolyse signature motif which harbours the catalytic serine residue in other FAE families. Six of the seven fae genes were successfully expressed heterologously in Escherichia coli, and the purified enzymes exhibited temperature optima range of 40-70 °C and the pH optima of between 6.5 and 8.0. The k(cat)/K(M) ratios for the six characterised FAEs showed the following order of substrate preference: methyl sinapate > methyl ferulate > ethyl ferulate. All six FAEs showed poor conversion rates against methyl p-coumarate and methyl caffeate, both of which lacked the methoxy (O-CH₃) group substituent on the aromatic ring of the ester substrates, emphasising the requirement for at least one methoxy group on the aromatic ring of the hydroxycinnamic acid ester substrate for optimal FAE activity.

Production, characterization and applications of organic solvent tolerant lipase by Pseudomonas aeruginosa AAU2

Abstract Jatropha seedcake (JSC) represents an important environmental problem owing to its toxicity for the biodiesel producing sectors where they are generated in bulk as a major by-product. The aim of the present work was to study the feasibility of JSC as a substrate for lipase production under submerged condition. Pseudomonas aeruginosa AAU2 isolated from dumped rotting JSC was found to produce extracellular solvent tolerant lipase (0.432 U ml −1 ). A 11.4-fold increase in enzyme yield was obtained in an optimized medium containing 20 g JSC/l, 2.5 g potassium nitrate/l and 0.1 v/v% Tween 80 after 60 h of incubation at 30 °C under shake flask conditions (150 rpm). The pH and temperature optima of the partially purified AAU2 lipase were 7.5 and 40 °C, respectively. The AAU2 lipase was found to be a cysteine hydrolase and exhibited preference towards longer carbon chain fatty acid ester substrates over the shorter ones. The enzyme exhibited significant stability in the presence of commercial detergents namely, Ezee ® and Wheel ® . The AAU2 lipase efficiently catalyzed biodiesel production using Jatropha oil. In addition, the enzyme also hydrolyzed 86.49% of commercially available phorbol ester (phorbol 12-myristate 13-acetate) within 12 h of incubation at 37 °C, suggesting its application in detoxification of phorbol ester from JSC. These results demonstrate viable approach for utilization of JSC biomass and degradation of phorbol esters by P. aeruginosa AAU2.