Bovine Brain Diacylglycerol Lipase: Substrate Specificity and Activation by Cyclic AMP-dependent Protein Kinase

Diacylglycerol lipase (EC 3.1.1.3) was purified from bovine brain microsomes using multiple column chromatographic techniques. The purified enzyme migrates as a single band on SDS-PAGE and has an apparent molecular weight of 27 kDa. Substrate specificity experiments using mixed molecular species of 1,2-diacyl-sn-glycerols indicate that low concentrations of Ca²⁺ and Mg²⁺ have no direct effect on enzymic activity and 1,2-diacyl-sn-glycerols are the preferred substrate over 1,3-diacyl-sn-glycerols. The enzyme hydrolyzes stearate in preference to palmitate from the sn-1 position of 1,2-diacyl-sn-glycerols. 1-O-Alkyl-2-acyl-sn-glycerols are not a substrate for the purified enzyme. The native enzyme had a V _max value of 616 nmol/min mg protein. Phosphorylation by cAMP-dependent protein kinase resulted in a threefold increase in catalytic throughput (V _max = 1,900 nmol/min mg protein). The substrate specificity and catalytic properties of the bovine brain diacylglycerol lipase suggest that diacylglycerol lipase may regulate protein kinase C activity and 2-arachidonoyl-sn-glycerol levels by rapidly altering the intracellular concentration of diacylglycerols.     

Modification of Substrate Specificity Resulting in an Epoxide Hydrolase with Shifted Enantiopreference for (2,3‐Epoxypropyl)benzene

Random mutagenesis targeted at hotspots of noncatalytic active‐site residues of potato epoxide hydrolase StEH1 combined with an enzyme‐activity screen allowed the isolation of enzyme variants displaying altered enantiopreference in the catalyzed hydrolysis of (2,3‐epoxypropyl)benzene. The wild‐type enzyme favored the S enantiomer with a ratio of 2.5:1, whereas the variant displaying the most radical functional changes showed a 15:1 preference for the R enantiomer. This mutant had accumulated four substitutions distributed over two out of four mutated hotspots: W106L, L109Y, V141K, and I151V. The underlying causes of the enantioselectivity were a decreased catalytic efficiency in the catalyzed hydrolysis of the S enantiomer combined with retained activity with the R enantiomer. The results demonstrate the feasibility of molding the stereoselectivity of this biocatalytically relevant enzyme.     

Effect of moisture content on immobilized lipase-catalyzed triacylglycerol hydrolysis under supercritical carbon dioxide flow in a tubular fixed-bed reactor

Surplus fats and oils were reacted with several lipases under supercritical fluid conditions for the purpose of obtaining value-added products. Lipases, however, require sufficient moisture content to act as effective biocatalysts. An immobilized lipase from Candida antarctica was chosen to examine the rate of enzyme moisture loss under laboratory ambient conditions and also under supercritical fluid conditions. A more important aspect was to determine the effect of lipase moisture content on the hydrolysis of triacylglycerols under the same supercritical fluid conditions. Under ambient conditions at constant air flow, the immobilized lipase lost water at the rate of 4 to 5%/h, from 48.3% moisture to a final moisture content of 0.2%. Water is known not to be very soluble in supercritical carbon dioxide (SC-CO₂). Nevertheless, under supercritical fluid conditions of 60°C, 4000 psi, and carbon dioxide flow rates of 0.5 or 1 L/min measured as expanded gas, the enzyme moisture loss was approximately 2 to 6%/h. To determine the effect of moisture loss on enzymatic hydrolysis, lipase beds in a tubular reactor with moisture contents of 1.5 to 23.5% were reacted with tripalmitin under supercritical conditions. A lipase with an initial moisture content of 1.5% gave little evidence of hydrolysis whereas those containing 5.4 to 23.5% moisture content resulted in products that contained only palmitic acid and unreacted tripalmitin. Thus, optimal parameters for continuous lipase hydrolysis of tripalmitin require: enough enzyme moisture to compensate for complete substrate hydrolysis; sufficient enzyme moisture for losses due to water solubility in SC-CO₂; temperature and pressure sufficient to solubilize the tripalmitin; high carbon dioxide total flow to solubilize all the tripalmitin; and a relatively large enzyme bed volume to increase the solubilized substrate contact time with the enzyme.     

Effect of Human Flavin-Containing Monooxygenase 3 Polymorphism on the Metabolism of Aurora Kinase Inhibitors

Aurora kinases were recently identified as a potential target in anticancer therapy and, amongst their available inhibitors, Tozasertib (VX-680) and Danusertib (PHA-739358) have been indicated as possible substrates of human flavin-containing monooxygenase 3 (hFMO3). Here we report the in vitro rate of oxidation of these drugs by wild-type hFMO3 and its polymorphic variant V257M. The conversion of Tozasertib and Danusertib to their corresponding metabolites, identified by LC-MS, by the purified wild-type and V257M hFMO3 show significant differences. In the case of Tozasertib, the V257M variant shows a catalytic efficiency, expressed as k_cat/K_m, similar to the wild-type: 0.39 ± 0.06 min⁻¹μM⁻¹ for V257M compared to 0.33 ± 0.04 min⁻¹μM⁻¹ for the wild type. On the other hand, in the case of Danusertib, V257M shows a 3.4× decrease in catalytic efficiency with k_cat/K_m values of 0.05 ± 0.01 min⁻¹μM⁻¹ for V257M and 0.17 ± 0.03 min⁻¹μM⁻¹ for the wild type. These data reveal how a simple V257M substitution ascribed to a single nucleotide polymorphism affects the N-oxidation of relevant anticancer drugs, with important outcome in their therapeutic effects. These findings demonstrate that codon 257 is important for activity of the hFMO3 gene and the codon change V to M has an effect on the catalytic efficiency of this enzyme.     

Trp89 in the lid ofHumicola lanuginosa lipase is important for efficient hydrolysis of tributyrin

To determine whether Trp89 located in the lid of the lipase (EC 3.1.1.3) fromHumicola lanuginosa is important for the catalytic property of the enzyme, site-directed mutagenesis at Trp89 was carried out. The kinetic properties of wild type and mutated enzymes were studied with tributyrin as substrate. Lipase variants in which Trp89 was changed to Phe, Leu, Gly or Glu all showed less than 14% of the activity compared to that of the wild type lipase. The Trp89Glu mutant was the least active with only 1% of the activity seen with the wild type enzyme. All Trp mutants had the same binding affinity to the tributyrin substrate interface as did the wild type enzyme. Wild type lipase showed saturation kinetics against tributyrin when activities were measured with mixed emulsions containing different proportions of tributyrin and the nonionic alkyl polyoxyethylene ether surfactant, Triton DF-16. Wild type enzyme showed a V_max=6000±300 mmol·min⁻¹·g⁻¹ and an apparent K_m=16±2% (vol/vol) for tributyrin in Triton DF-16, while the mutants did not show saturation kinetics in an identical assay. The apparent K_m for tributyrin in Triton DF-16 was increased as the result of replacing Trp89 with other residues (Phe, Leu, Gly or Glu). The activities of all mutants were more sensitive to the presence of Triton DF-16 in the tributyrin substrate than was wild type lipase. The activity of the Trp89Glu mutant was decreased to 50% in the presence of 2 vol% Triton DF-16 compared to the activity seen with pure tributyrin as substrate. Wild type lipase and all mutants except Trp89Glu had the same affinity for the substrate interface formed by 15.6 vol% tributyrin in Triton DF-16. The Trp89Glu mutant showed a lower affinity than all the other lipase variants for the interface of 15.6 vol% tributyrin in Triton DF-16. The study showed that Trp89 located in the lid ofH. lanuginosa lipase is important for the efficient hydrolysis of tributyrin and that this residue plays a role in the catalytic steps after adsorption of the lipase to the substrate interface.     

Engineering of Pseudomonas aeruginosa lipase by directed evolution for enhanced amidase activity: mechanistic implication for amide hydrolysis by serine hydrolases

A lipase from Pseudomonas aeruginosa was subjected to directed evolution for increased amidase activity to probe the catalytic mechanism of serine hydrolases for the hydrolysis of amides. Random mutagenesis combined with saturation mutagenesis for all the amino acid residues at the substrate-binding site successfully identified the mutation at the residue 252 next to the catalytic H251 as a hot spot for selectively increasing the amidase activity of the lipase. The saturation mutagenesis targeted for the oxyanion hole (M16 and H83) gave no positive results. The substitutions of Met or Phe for Leu252 significantly increased the amidase activity toward N-(2-naphthyl)oleamide (2), whereas the esterase activity toward structurally similar 2-naphthyl oleate (1) was not affected by the substitution. The triple mutant F207S/A213D/M252F (Sat252) exhibited amidase activity (k(cat)/K(m)) 28-fold higher than that of the wild-type lipase. Kinetic analysis of Sat252 and its parental clone 10F12 revealed that the amidase activity was increased by the increase in the catalytic efficiency (k(cat)). The increase in k(cat) suggested the importance of the leaving group protonation by the catalytic His during the break down of the tetrahedral intermediate in the hydrolysis of amides.     

Engineering cytochrome P-450cam to increase the stereospecificity and coupling of aliphatic hydroxylation

Site-directed mutants were constructed in cytochrome P-450_camto re-engineer the stereochemistry and coupling of ethylbenzenehydroxyiation. The reaction with wild-type (WT) enzyme producesone regioisomer 1-phenylethanol with 5% reduced nicotinamideadenine deoxyribonucleic acid to product conversion of and aratio of 73:27 for the R and S enantiomers respectively. Ethyibenzenewas modeled into the active site of WT P-450_cam in a rigid modeand oriented to optimize either pro-R or pro-S hydrogen abstraction.Residues T101, T185 and V247 make extensive contacts with thesubstrate in the static complexes and were therefore chosenfor site-directed mutagenesis. Single mutants T101M, V247A andV247M are more stereospedik producing 89,87 and 82% (R)-1-phenylethanolrespectively. The coupling of the reaction is doubled for thesingle mutants T185L, T185F and V247M. In an effort to engineerincreased stereospecificity and coupling into a single catalystthe T101M, T185F and V247M mutants were combined in a multiplemutant of P-450_cam.This protein hydroxylates ethyibenzene resultingin an R:S ratio of 87:13 for the 1-phenylethanols and 13% couplingof reducing equivalents to product. The catalytic stereospecificityand stoichiometry with T101M–T185F–V247M does notrepresent a summation of the changes observed for the singlemutants. A portion of the individual effects on substrate recognitionproduced by the single substitutions is either eliminated ordegenerate within the triple mutant.     

Lipase-catalyzed hydrolysis of canola oil in supercritical carbon dioxide

The effect of pressure, temperature, and CO₂ flow rale on the extent of conversion and the product composition in the enzyme-catalyzed hydrolysis of canola oil in supercritical carbon dioxide (SCCO₂) was investigated using lipase from Mucor miehei immobilized on macroporous anionic resin (Lipozyme IM). Reactions were carried out in a continuous flow reactor at 10, 24, and 38 MPa and 35 and 55°C. Supercritical fluid chromatography was used to analyze the reaction products. A conversion of 63–67% (triglyceride disappearance) was obtained at 24–38 MPa. Mono-and diglyceride production was minimum at 10 MPa and 35°C. Monoglyceride production was favored at 24 MPa. The amount of product obtained was higher at 24–38 MPa due to enhanced solubility in SCCO₂. Complete hydrolysis of oil should be possible by increasing the enzyme load and/or decreasing the quantity of the oil substrate. There was a drop in triglyceride conversion over a 24-h reaction time at 38 MPa and 55°C, which may be an indication of loss of enzyme activity. Pressure, temperature, and CO₂ flow rate are important parameters to be optimized in the enzyme-catalyzed hydrolysis of canola oil in SCCO₂ to maximize its conversion to high-value products.     

Protein engineering and applications of <Emphasis Type="Italic">Candida rugosa</Emphasis> lipase isoforms

Commercial preparations of Candida rugosa lipase (CRL) are mixtures of lipase isoforms used for the hydrolysis and synthesis of various esters. The presence of variable isoforms and the amount of lipolytic protein in the crude lipase preparations lead to a lack of reproducibility of biocatalytic reactions. Purification of crude CRL improve their substrate specificity, enantioselectivity, stability, and specific activities. The expression of the isoforms is governed by culture or fermentation conditions. Unfortunately, the nonsporogenic yeast C. rugosa does not utilize the universal codon CTG for leucine; therefore, most of the CTG codons were converted to universal serine triplets by site-directed mutagenesis to gain expression of functional lipase in heterologous hosts. Recombinant expressions by multiple-site mutagenesis or complete synthesis of the lipase gene are other possible ways of obtaining pure and different CRL isoforms, in addition to culture engineering. Protein engineering of purified CRL isoforms allows the tailoring of enzyme function. This involves computer modeling based on available 3-D structures of lipase isoforms. Lid swapping and DNA shuffling techniques can be used to improve the enantioselectivity, thermostability, and substrate specificity of CRL isoforms and increase their biotechnological applications. Lid swapping can result in chimera proteins with new functions. The sequence of the lid can affect the activity and specificity of recombinant CRL isoforms. Candida rugosa lipase is toxicologically safe for food applications. Protein engineering through lid swapping and rationally designed site-directed mutagenesis will continue to lead to the production of CRL isoforms with improved catalytic power, thermostability, enantioselectivity, and substrate specificity, while providing evidence for the mechanisms of actions of the various isoforms.     

Protein Engineering of the Antitumor Enzyme PpADI for Improved Thermal Resistance

Arginine deiminase (ADI, EC 3.5.3.6) is a potential antitumor drug for the treatment of arginine‐auxotrophic tumors such as hepatocellular carcinomas (HCCs) and melanomas. Studies in human lymphatic leukemia cell lines have confirmed the anti‐angiogenic activity of ADI. Activity and thermal resistance limit the efficacy of ADI in treatment of auxotrophic tumors. Previously, we reengineered ADI from Pseudomonas plecoglossicida (PpADI) for improved activity under physiological conditions (37 °C, PBS buffer, pH 7.4) by two rounds of directed evolution and combination of beneficial substitutions through site‐directed mutagenesis. The best variant, PpADI M6 (K5T/D38H/D44E/A128T/E296K/H404R), showed a 64.7‐fold improvement in k_cat value and a 37.6 % decreased S_0.5 value under physiological conditions. However, M6 lost rapidly its activity (half‐life of ～2 days at 37 °C). Here we report the re‐engineering of PpADI M6 for improved thermal resistance by directed evolution in order to increase its half‐life under physiological conditions. Directed evolution and recombination of the two most beneficial positions yielded variant PpADI M9 (K5T/D38H/D44E/A128T/V140L/E296K/F325L/H404R), for which the T_m value increased from 47 (M6) to 54 °C (M9); this corresponds to an increased half‐life from ～2 days (M6) to ～3.5 days (M9) under physiological conditions. Structure analysis of the homology model of M9 showed that the beneficial substitutions V140L and F325L likely promote the formation of tetrameric PpADI, which has greater thermal resistance than dimeric PpADI.     

Directed evolution of subtilisin E into a highly active and guanidinium chloride- and sodium dodecylsulfate-tolerant protease

Proteases have niche applications in diagnostic kits that use cell lysis and thereby require high resistance towards chaotropic salts and detergents, such as guanidinium chloride (GdmCl) and sodium dodecylsulfate (SDS). Subtilisin E, a well‐studied serine protease, was selected to be re‐engineered by directed evolution into a “chaophilic” protease that would be resistance to GdmCl and SDS, for application in diagnostic kits. In three iterative rounds of directed evolution, variant SeSaM1–5 (S62I/A153V/G166S/I205V) was generated, with improved activity (330 %) and increased half life in 1 M GdmCl (<2 min to 4.7 h) or in 0.5 % SDS (<2 min to 2.7 h). Saturation mutagenesis at each site in the wild‐type subtilisin E revealed that positions 62 and 166 were mainly responsible for increased activity and stability. A double mutant, M2 (S62I/G166M), generated by combination of the best single mutations showed significantly improved kinetic constants; in 2 M GdmCl the K_m value decreased (29‐fold) from 7.31 to 0.25 mM , and the k_cat values increased (fourfold) from 15 to 61 s^?1. The catalytic efficiency, k_cat/K_m, improved dramatically (GdmCl: 247 mM ^?1 s^?1 (118‐fold); SDS, 179 mM ^?1 s^?1 (13‐fold)). In addition, the SeSaM1–5 variant showed higher stability in 2.0 % SDS when compared to the wild‐type (t_1/2 54.8 min (>27‐fold)). Finally, molecular dynamics simulations of the wild‐type subtilisin E showed that Gdm⁺ ions could directly interact with active site residues, thereby probably limiting access of the substrate to the catalytic centre.     

Residue Asn277 Affects the Stability and Substrate Specificity of the SMG1 Lipase from Malassezia globosa

Thermostability and substrate specificity are important characteristics of enzymes for industrial application, which can be improved by protein engineering. SMG1 lipase from Malassezia globosa is a mono- and diacylglycerol lipase (MDL) that shows activity toward mono- and diacylglycerols, but no activity toward triacylglycerols. SMG1 lipase is considered a potential biocatalyst applied in oil/fat modification and its crystal structure revealed that an interesting residue-Asn277 may contribute to stabilize loop 273–278 and the 3₁₀4 helix which are important to enzyme characterization. In this study, to explore its role in affecting the stability and catalytic activity, mutagenesis of N277 with Asp (D), Val (V), Leu (L) and Phe (F) was conducted. Circular dichroism (CD) spectral analysis and half-life measurement showed that the N277D mutant has better thermostability. The melting temperature and half-life of the N277D mutant were 56.6 °C and 187 min, respectively, while that was 54.6 °C and 121 min for SMG1 wild type (WT). Biochemical characterization of SMG1 mutants were carried out to test whether catalytic properties were affected by mutagenesis. N277D had similar enzymatic properties as SMG1 WT, but N277F showed a different substrate selectivity profile as compared to other SMG1 mutants. Analysis of the SMG1 3D model suggested that N277D formed a salt bridge via its negative charged carboxyl group with a positively charged guanidino group of R227, which might contribute to confer N277D higher temperature stability. These findings not only provide some clues to understand the molecular basis of the lipase structure/function relationship but also lay the framework for engineering suitable MDL lipases for industrial applications.     

Acidolysis of Tripalmitin with Oleic Acid Catalyzed by a Newly Isolated Thermostable Lipase

The catalytic efficiency of a lipase from Bacillus stearothermophilus MC7 (lipase MC7) was evaluated in acidolysis of tripalmitin with oleic acid to yield dioleoylpalmitoylglycerol, a structured triglyceride used in health food. The immobilized enzyme exhibits good operational thermostability with a half-life of 50 days at 60 °C in a solvent-free system. The degree of conversion exceeded 50% after 48 h. The side reaction of hydrolysis was suppressed. However, the monosubstituted product was prevalent in the product mixture. Tested in a broad range of solvents, lipase MC7 showed tolerance towards medium polarity.     

Catalyst design using artificial intelligence: SO2 to SO3 case study

Catalyst design is key to the improvement of chemical process efficiency. The required work for the development of new catalysts can be supported through the proper application of artificial intelligence to identify optimal compositions. A generic methodology for the application of machine learning to catalysis research is therefore outlined in this work. The catalytic oxidation of SO₂ was used to exemplify the first iteration of this methodology. 1784 data points from 31 published papers were compiled into a databank. The inlet SO₂ concentration ranged from 0 to 66 mol%. An artificial neural network (ANN) was trained on the databank in order to predict SO₂ conversion based on the catalyst composition and the reactor operating conditions (temperature, pressure, catalyst mass: volumetric flowrate ratio (w/v), and feed composition). The model achieved a root-mean-square error of 6.6%. A preliminary screening step identified 3:1 V-Mg/SiO₂ catalysts as exhibiting high conversion at 648 K. A multi-objective optimization was then performed on a single catalyst to identify solutions exhibiting high conversion and high productivity at 648 K while minimizing the catalyst cost. The optimal solution was predicted to be a 2.9 wt% V/0.2 wt% Mg/SiO₂ catalyst operating at a w/v of 7.49 kg-cat · s/m³ STP, achieving 100% SO₂ conversion with a material cost among the bottom third of cost values. Artificial intelligence can then be employed to extract useful knowledge from published catalytic data and orient future search for novel catalyst development.     

Catalytic deNO x properties of novel vanadium oxide based open-framework materials

The deNO _x catalytic properties of a new class of open-framework structure materials, Li₆[Mn₃(H₂O)₁₂V₁₈O₄₂(XO₄)] · 24H₂O (X = V, S) (1), [Fe₃(H₂O)₁₂ V₁₈O₄₂(XO₄)] · 24H₂O (X = V, S) (2), [Co₃(H₂O)₁₂V₁₈O₄₂(XO₄)] · 24H₂O (X = V, S) (3), and Li₆[Ni ₃ ^II (H₂O)₁₂V ₁₆ ^VI V ₂ ^V O₄₂(SO₄)] · 24H₂O (4), have been studied. The crystal structures of these novel systems consist of three-dimensional arrays of vanadium oxide clusters {V₁₈O₄₂(XO₄)} , as building block units, interlinked by {–O–M–O–} (M = Mn, 1; M = Fe, 2; M = Co, 3; M = Ni, 4) bridges. Their open-framework structures contain cavities, similar to those observed in conventional zeolites, which are occupied by exchangeable cations and/or readily removable water of hydration. The catalysts derived from these materials were tested for the selective catalytic reduction (SCR) of nitrogen oxides {NO _x } into N₂ using a hydrocarbon, propylene, as the reducing agent. The catalysts were ineffective under lean burn conditions. However, the new catalysts, especially the one derived from the cobalt derivative (3), showed intriguing deNO _x activity under rich conditions. They remove up to ~ 99% of the toxic NO _x emissions in 1.5% O₂ with 100% selectivity to N₂. The active phase of the catalysts exhibit good stability, can be readily regenerated, and are selective to the desired product-N₂. The catalytic reactions occur at moderately low temperatures (400–500 °C). The catalysts were characterized by FT-IR, temperature programmed reactions (TPR and TPO), SEM, BET surface area measurements, elemental analysis, and X-ray diffraction (XRD). Additional advanced techniques were used to further characterize the catalyst phases that showed most promising deNO _x activity and increased tolerance to oxygen.     

The triglyceride composition,structure, and presence of estolides in the oils ofLesquerella and related species

Members of the genusLesquerella, native to North America, have oils containing large amounts of hydroxy fatty acids and are under investigation as potential new crops. The triglyceride structure of oils from twenty-fiveLesquerella species in the seed collection at our research center has been examined after being hydrolysis-catalyzed by reverse micellar-encapsulated lipase and alcoholysis-catalyzed by immobilized lipase. These reactions, when coupled with supercritical-fluid chromatographic analysis, provide a powerful, labor-saving method for oil triglyceride analysis. A comprehensive analysis of overall fatty acid composition of these oils has been conducted as well.Lesquerella oils (along with oils from two other Brassicaceae:Physaria floribunda andHeliophilia amplexicaulis) have been grouped into five categories: densipolic acid-rich (Class I); auricolic acid-rich (Class II); lesquerolic acid-rich (Class III); an oil containing a mixture of hydroxy acids (Class IV); and lesquerolic and erucic acid-rich (Class V). The majority of Class I and II triglycerides contain one or two monoestolides at the 1- and 3-glycerol positions and a C₁₈ polyunsaturated acyl group at the 2-position. Most Class III and IV oil triglycerides contain one or two hydroxy acids at the 1- and 3-positions and C₁₈ unsaturated acid at the 2-position. A few of the Class III oils have trace amounts of estolides. The Class V oil triglycerides are mostly pentaacyl triglycerides and contain monestolide and small amounts of diestolide. Our triglyceride structure assignments were supported by¹H nuclear magnetic resonance data and mass balances.     

Alteration of substrate specificity of cholesterol oxidase from Streptomyces sp. by site-directed mutagenesis

Despite the structural similarities between cholesterol oxidasefrom Streptomyces and that from Brevibacterium, both enzymesexhibit different characteristics, such as catalytic activity,optimum pH and temperature. In attempts to define the molecularbasis of differences in catalytic activity or stability, substitutionsat six amino acid residues were introduced into cholesteroloxidase using site-directed mutagenesis of its gene. The aminoacid substitutions chosen were based on structural comparisonsof cholesterol oxidases from Streptomyces and Brevibacterium.Seven mutant enzymes were constructed with the following aminoacid substitutions: L117P, L119A, L119F, V145Q, Q286R, P357Nand S379T. All the mutant enzymes exhibited activity with theexception of that with the L117P mutation. The resulting V145Qmutant enzyme has low activities for all substrates examinedand the S379T mutant enzyme showed markedly altered substratespecificity compared with the wild-type enzyme. To evaluatethe role of V145 and S379 residues in the reaction, mutantswith two additional substitutions in V145 and four in S379 wereconstructed. The mutant enzymes created by the replacement ofV145 by Asp and Glu had much lower catalytic efficiency forcholesterol and pregnenolone as substrates than the wild-typeenzyme. From previous studies and this study, the V145 residueseems to be important for the stability and substrate bindingof the cholesterol oxidase. In contrast, the catalytic efficiencies(k_cat/K_m) of the S379T mutant enzyme for cholesterol and pregnenolonewere 1.8- and 6.0-fold higher, respectively, than those of thewild-type enzyme. The enhanced catalytic efficiency of the S379Tmutant enzyme for pregnenolone was due to a slightly high k_catvalue and a low K_m value. These findings will provide severalideas for the design of more powerful enzymes that can be appliedto clinical determination of serum cholesterol levels and assterol probes.     

Analysis of volume‐to‐surface ratio effects on methane oxidative coupling using microkinetic modeling

The effect of the volume‐to‐surface (V/S) ratio on the catalytic performance of a La–Sr/CaO catalyst in a fixed bed reactor under oxidative coupling of methane (OCM) conditions is investigated by adjusting the amount of diluent in the catalyst bed. It was observed experimentally that the catalyst activity, C₂ selectivity, and C₂H₄/C₂H₆ ratio are all favored at high V/S ratios. The total void volume, available in the intraparticle and the interstitial phase, was considered. A comprehensive OCM microkinetic model, explicitly distinguishing between these two phases, allowed accounting for the observed dependence of catalytic performance on V/S ratio. The major experimentally implemented variation in interstitial volume available for reaction, provoked also changes in radical concentration profiles in intraparticle phase. Given the high reaction rates occurring at this location, the experimentally observed effects with varying the V/S ratio, are attributed to concentration and, hence, reaction rate changes occurring mainly in the intraparticle phase. © 2018 American Institute of Chemical Engineers AIChE J, 64: 2603–2611, 2018     

Long-term stability of the V2O5/Al2O3 catalyst for the selective reduction of nitrogen oxides

Changes of the V₂O₅/Al₃O₃ catalyst aged for up to 10 years under real conditions of the selective catalytic reduction of NO _x by ammonia (SCR) at the tail gases of the nitric acid plant were characterized by⁵¹V NMR spectroscopy, porosimetry, temperature programmed reduction (TPR) and catalytic activity measurements. The catalytic activity and the redox properties of the catalyst were found intact. Only small variations of the ratio of the octahedral and tetrahedral vanadia species were documented by⁵¹V NMR on aged catalyst.     

Conversion of human 15-lipoxygenase to an efficient 12-lipoxygenase: the side-chain geometry of amino acids 417 and 418 determine positional specificity

Positional specificity determinants of human 15-lipoxygenasewere examined by site-directed mutagenesis and by kinetic analysisof the wild-type and variant enzymes. By comparing conserveddifferences among sequences of 12- and 15-lipoxygenases, a smallregion responsible for functional differences between 12- and15-lipoxygenases has been identified. Furthermore, the replacementof only two amino acids in 15-lipoxygenase (at 417 and 418 inthe primary sequence) by those found in certain 12-lipoxygenasesresults in an enzyme that has activity similar to 12-lipoxygenase.An examination of the activity of nine variants of lipoxygenasedemonstrated that the amino acid side-chain bulk and geometryof residues 417 and 418 are the key components of the positionalspecificity determinant of 15-lipoxygenase. Overexpression ofa variant (containing valines at positions 417 and 418) thatperforms predominantly 12-lipoxygenation was achieved in a baculovirus-insectcell culture system. This variant was purified to >90% homogeneityand its kinetics were compared with the wild-type 15-lipoxygenase.The variant enzyme has no change in its apparent K_M for arachidonicacid and a minor(3-fold) change in its V_max. For linoleic acid,the variant has no change in its K_M and a 10-fold reductionin its V_max, as expected for an enzyme performing predominantly12-lipoxygenation. The results are consistent with a model inwhich two amino acids of 15-lipoxygenase (isoleucine 417 andmethionine 418) constitute a structural element which contributesto the regiospecificity of the enzyme. Replacement of theseamino acids with those found in certain 12-lipoxygenases resultsin an enzyme which can bind arachidonic acid in a catalyticregister that prefers 12-lipoxygenation.