Mutagenesis of an Asn156 Residue in a Surface Region of S‐Selective Hydroxynitrile Lyase from Baliospermum montanum Enhances Catalytic Efficiency and Enantioselectivity

The S‐selective hydroxynitrile lyase from Baliospermum montanum (BmHNL) has broad substrate specificity toward aromatic substrates as well as high temperature stability, although with low enantioselectivity and specific activity. To expand the industrial application of this enzyme, we improved its enantioselectivity and specific activity toward (S)‐mandelonitrile by mutagenesis. The specific activity of the BmHNL H103C/N156G mutant for (S)‐mandelonitrile production was raised to 154 U mg^?1 (WT BmHNL: 52 U mg^?1). The enantiomeric excess was increased to 93 % (WT BmHNL: 55 %). The kinetic analysis revealed K_m for (R)‐mandelonitrile and k_cat for (S)‐mandelonitrile increased by the mutation at Asn156, thus contributing to the increase in enantiomeric excess. This is the first report on an improvement in catalytic efficiency and enantiomeric excess of BmHNL for (S)‐mandelonitrile synthesis by random and site‐directed mutagenesis.     

Resin Acid Conversion with CYP105A1: An Enzyme with Potential for the Production of Pharmaceutically Relevant Diterpenoids

Cytochrome P450s are very versatile enzymes with great potential for biotechnological applications because of their ability to oxidize unactivated C? H bonds. CYP105A1 from Streptomyces griseolus was first described as a herbicide‐inducible sulfonylurea hydroxylase, but it is also able to convert other substrates such as vitamin D₃. To extend the substrate pool of this interesting enzyme further, we screened a small diterpenoid compound library and were able to show the conversion of several resin acids. Binding of abietic acid, dehydroabietic acid, and isopimaric acid to the active site was assayed, and V_max and K_m values were calculated. The products were analyzed by NMR spectroscopy and identified as 15‐hydroxyabietic acid, 15‐hydroxydehydroabietic acid, and 15,16‐epoxyisopimaric acid. As the observed products are difficult to obtain by chemical synthesis, CYP105A1 has proved to be a promising candidate for biotechnological applications that combine bioconversion and chemical synthesis to obtain functionalized resin acids.     

Alzheimer’s Disease: Identification and Development of β‐Secretase (BACE‐1) Binding Fragments and Inhibitors by Dynamic Ligation Screening (DLS)

The application of dynamic ligation screening (DLS), a methodology for fragment‐based drug discovery (FBDD), to the aspartic protease β‐secretase (BACE‐1) is reported. For this purpose, three new fluorescence resonance energy transfer (FRET) substrates were designed and synthesized. Their kinetic parameters (V_max, K_M, and k_cat) were determined and compared with a commercial substrate. Secondly, a peptide aldehyde was designed as a chemically reactive inhibitor (CRI) based on the Swedish mutation substrate sequence. Incubation of this CRI with the protease, a FRET substrate, and one amine per well taken from an amine library, which was assembled by a maximum common substructure (MCS) approach, revealed the fragment 3‐(3‐aminophenyl)‐2H‐chromen‐2‐one ( 1 ) to be a competitive BACE‐1 inhibitor that enhanced the activity of the CRI. Irreversibly formed fragment combination products of 1 with the initial peptide sequence were active and confirmed the targeting of the active site through the ethane‐1,2‐diamine isostere. Finally, structure‐assisted combination of fragment 1 with secondary fragments that target the S1 site in hit optimization yielded novel, entirely fragment‐based BACE‐1 inhibitors with up to 30‐fold improved binding affinity. Interactions with the protein were explained by molecular modeling studies, which indicate that the new fragment combinations interact with the catalytic aspartic acid dyad, as well as with the adjacent binding sites required for potency.     

Determination of the phospholipase activity of patatin by a continuous spectrophotometric assay

Patatin is a family of glycoproteins that accounts for 30–40% of the total soluble protein in potato (Solanum tuberosum L.) tubers. This protein has been reported to serve as a storage protein and also to exhibit lipid phospholipase activity. This paper describes a simple continuous spectrophotometric method for assaying patatin phospholipase activity. The procedure is based on a coupled enzymatic assay using [1,2-dilinoleoyl]PC as the phospholipase substrate and lipoxygenase as the coupling enzyme. In the procedure developed in this work, lipoxygenase oxidizes the linoleic acid released by the phospholipase activity of patatin. This activity can then be followed spectrophotometrically by recording the increase in absorbance at 234 nm that results from the formation of the corresponding hydroperoxide from linoleic acid by the action of lipoxygenase. The optimal assay concentrations of patatin and lipoxygenase were established. Phospholipase activity varied with pH, reaching its optimal value at pH 9.5. Scans of the deoxycholate concentration pointed to an optimal detergent concentration of 3 mM. Phospholipid hydrolysis followed classical Michaelis-Menten kinetics (V _m=9.8×10⁻³ μmol/min·μg protein, K _m=7.8 μM, V _m/K _m=1.3 min⁻¹·μg protein). This method proved to be specific since there was no activity in the absence of patatin. It also had the advantages of a short analysis time and the use of commercially nonradiolabeled and inexpensive substrates, which are, furthermore, natural substrates of phospholipase.     

Attempts to synthesize the framework nitrogen bearing zeolites

The attempts to synthesize the nitrogen bearing zeolite analogs have been conducted under conditions resembling hydrothermal synthesis of zeolite with SiCl₄, AlCl₃, AlN, or metallic Al, and NaNH₂, as principal substrates. Ammonolysis of the substrates (mostly SiCl₄) was always the first step of synthesis and yielded the amorphous, porous products showing very high surface area and well ordered mesopore system. Their further treatment with NaNH₂ in liquid NH₃ or in other aprotic solvents (i.e., crystallization) usually did not lead to crystalline, porous materials, whereas the porosity of the products declined markedly. The crystalline products have been obtained by ammonolysis of Al₂(SiF₆)₃, but the product showed a low thermal stability.     

The use of a specific function to estimate maximum methane production in a batch‐fed anaerobic reactor

A readily biodegradable substrate was used to assess the value of using a mathematical function of y = y_maxexp^m/x as a simplified method of determining the maximum methane production (G_max) in a batch anaerobic reactor. Experimental results to test the method used three different initial substrate loadings in pre‐acclimatised completely mixed anaerobic reactors. Gas production was found to follow a typical trend that has previously been described by first order reaction kinetics; for the purpose of fitting the linearisation, it requires a value for maximum cumulative methane production. Use of the modified specific function to yield the equation G = G_maxexp^m/t showed that the experimental gas production curve could be estimated with a high degree of similarity. This was confirmed by a statistical analysis using the method of residuals which gave a correlation coefficient (R²) greater than 0.97 between experimental and estimated values. Using a graphical linearisation of the specific function produced a simplified method of predicting G_max. The value obtained was then used in a first order kinetic model to derive the specific coefficient rate (K_o), which was in agreement with other methods used for its determination. Copyright © 2004 Society of Chemical Industry     

Kinetic data and substrate specificity of a keratinase from Chryseobacterium sp. strain kr6

BACKGROUND: Keratinases are important enzymes for biotechnological processes involving keratin hydrolysis. In this work substrate specificity and kinetic properties of a keratinase from Chryseobaterium sp. were investigated. RESULTS: The optimal conditions for activity of purified keratinase with respect to pH, temperature and sodium chloride concentration were established using factorial design and surface response techniques. The optimum conditions for keratinase activity were pH from 7.4 to 9.2, temperature from 35 °C to 50 °C and NaCl concentration from 50 to 340 mmol L^?1, having azocasein as substrate. Subsequently, the kinetic parameters for this substrate were determined to be K_m = 0.75 mg mL^?1 and V_max = 59.5 U min^?1. The K_i value for 1,10‐phenanthroline was estimated at 0.78 mmol L^?1. The enzyme specificity was evaluated over different synthetic and insoluble substrates. The protease exhibited specificity with selectivity for hydrophobic and positively charged residues. In relation to the insoluble substrates, the enzyme hydrolyzed preferably chicken nails. CONCLUSIONS: This enzyme effectively hydrolyzes insoluble keratin substrates. The knowledge of keratinase properties is an essential step in the development of biotechnological processes involving keratin hydrolysis. Copyright © 2008 Society of Chemical Industry     

Synthesis and Characterization of Pyridine Compounds for Amperometric Measurements of Leukocyte Esterase

We introduce a new class of substrates (compounds I – III ) for leukocyte esterase (LE) that react with LE yielding anodic current in direct proportion to LE activity. The kinetic constants K_m and k_cat for the enzymatic reactions were determined by amperometry at a glassy carbon electrode. The binding affinity of I – III for LE was two orders of magnitude better than that of existing optical LE substrates. The specificity constant k_cat/K_m was equal to 2.7, 3.8, and 5.8×10⁵ m ^?1 s^?1 for compounds containing the pyridine ( I ), methoxypyridine ( II ), and (methoxycarbonyl)pyridine ( III ), respectively, thus showing an increase in catalytic efficiency in this order. Compound III had the lowest octanol/water partition coefficient (log p=0.33) along with the highest topological surface area (tPSA=222 Ų) and the best aqueous solubility (4.0 mg mL^?1). The average enzymatic activity of LE released from a single leukocyte was equal to 4.5 nU when measured with compound III .     

Pyrolysis of propane at reflected shock-wave temperatures from 1300 to 2700 K

Shock tube pyrolysis of propane at temperatures between 1300 K and 2700 K at reflected shock pressures of 500 to 1500 kN/m² has been investigated. The reaction is of 1st order with a rate constant K = 1.79 × 10⁸ exp (-176.2 kJ/RT) s^?1. The major reaction products were acetylene, ethylene and methane, while traces of propylene and ethane were only detected at temperatures below 1500 K. At higher temperatures, propane conversion to acetylene increased at the expense of the other products. Optimum conversions to ethylene and methane, in contrast to that to acetylene, were more sensitive to changes in temperature than to variations in reaction time. However, at reaction pressures above 550 kN/m², extension of reaction time beyond 0.5 ms did not favour the formation of acetylene. A simple kinetic model which confirmed the experimental optimum product selectivity conditions is put forward.     

Lignin peroxidase‐catalyzed polymerization and detoxification of toxic halogenated phenols

Halogenated phenols and bisphenols are recognized as being recalcitrant in conventional biological treatments. The current research evaluated lignin peroxidase‐catalyzed oxidation and polymerization as a potential alternative for their detoxification. Gel permeation–HPLC analysis demonstrated the formation of dimers, trimers and tetramers upon oxidation of the target substrates. Polymerization was accompanied by effective detoxification of the aqueous phase during oxidation of 2,4‐dibromophenol, the extent of which correlated with the extent of oxidation and polymerization. Steady state kinetic measurements at a saturating concentration of H₂O₂ revealed high K_m values (270–1100 µmol dm^?3) for the target substrates, reflecting the strong electron‐withdrawing properties of halogen substituents, which increase the oxidation potential of the phenols, resulting in thermodynamically less favorable reactions. However, k_cat values were not dissimilar from non‐halogenated phenols and the rapid oxidation and polymerization suggests that low retention times could be expected in a continuous process for their treatment, in contrast to conventional biological methods. The operational stability of lignin peroxidase was significantly improved by inclusion of redox mediators, which resulted in enhanced oxidation and more rapid reaction rates. However, due to their inherent toxicity, the use of redox mediators impeded toxicity assays. The findings highlight the potential of lignin peroxidase as a possible alternative for the high‐rate treatment of industrial wastewater when conventional methods are ineffective. Copyright © 2003 Society of Chemical Industry     

Metabolic responses of microorganisms growing on inhibitory substrates in nonsteady state culture

The dynamics of metabolic activity of Pseudomonas putida cultured on phenol in a nonsteady state, resulting from simultaneous step increases (or decreases) in dilution rate and feed phenol concentration, were studied. Metabolic activity was determined by the uptake rate, defined as the mass of phenol degraded by a unit mass of bacteria in unit time. A minimum duration of nonsteady state was necessary to induce the metabolic pathway. The induction time for Pseudomonas putida cultured on phenol was approximately 30 min, irrespective of the type of disturbance applied. The Monod-Haldane equation was applied to fit the experimental values of uptake rate and the best kinetic parameters were calculated by the nonlinear least squares technique. Values of the kinetic parameters differed both for each parameter and type of disturbance investigated. For simultaneous step increases in dilution rate and feed phenol concentration, the value of u_m in the transient state first increased monotonically approximately four times and then decreased monotonically to the new steady state value after three residence times. Values of K_s and K_i attained those at the new steady state after three residence times, the highest being approximately three times greater than those of the steady state prior to disturbance. For simultaneous step decreases in D and S_O, values of u_m, K_s and K_i reached those at the new steady state values after four residence times.     

New Insights into the Biosynthesis of Prenylated Xanthones: Xptb from Aspergillus nidulans Catalyses an O‐Prenylation of Xanthones

Gene‐inactivation experiments have indicated that the putative prenyltransferase XptB from Aspergillus nidulans was likely to be responsible for the prenylation of 1,7‐dihydroxy‐6‐methyl‐8‐hydroxymethylxanthone. Recently, it was suggested that this enzyme might also accept as substrate the benzophenone arugosin H, which is assumed to be a precursor of prenylated xanthones. In this study, five benzophenones and ten xanthones were incubated with purified recombinant XptB in the presence of dimethylallyl diphosphate (DMAPP). XptB accepted four xanthones as substrates, including the proposed natural substrate, and catalysed regiospecific O‐prenylations at C‐7 of the xanthone core. K_m values in the range of 0.081–1.1 mM and turnover numbers (k_cat) between 0.02 and 0.5 s^?1 were determined for the accepted xanthones. The kinetic parameters for DMAPP were found to be 0.024 mM (K_m) and 0.13 s^?1 (k_cat). Arugosin H was not accepted by XptB under the tested conditions. XptB was relatively specific towards its prenyl donor and did not accept geranyl or farnesyl diphosphate as substrate. Mn²⁺ and Co²⁺ strongly enhanced XptB activity (up to eightfold); this has not been reported before for prenyltransferases of the DMATS superfamily.     

Effect of diffusional resistances on the action pattern of immobilized alpha-amylase

Alpha-amylase from Aspergillus oryzae has been immobilized onto corn grits and porous silica (specific areas 180 and 440 m² g^?1). Kinetic parameters of immobilized enzyme have been determined. Immobilization of alpha-amylase results in the formation of less polymerized products resulting in an apparent decrease in the number of transglycosylation reactions, for both maltotetraose and starch as substrates, when compared with free enzyme. Diffusional limitations for substrate and products have been quantified in the case of the three supports used. External diffusional resistances were important in all cases for the reaction products, whilst they became negligible for the substrate in the case of silica supports. Moreover, internal transfer limitations were identified with silica 180 m²g^?1 support. It was demonstrated that diffusional resistances were in direct relation to the apparent modification of the enzyme action pattern after immobilization.     

Chlorophyllide a oxidoreductase Preferentially Catalyzes 8-Vinyl Reduction over B-Ring Reduction of 8-Vinyl Chlorophyllide a in the Late Steps of Bacteriochlorophyll Biosynthesis

Bacteriochlorophyll a (BChl) is an essential pigment for anoxygenic photosynthesis. In late steps of the BChl biosynthesis of Rhodobacter capsulatus, the C8 vinyl group and C7=C8 double bond of 8-vinyl chlorophyllide a (8 V-Chlide) are reduced by a C8 vinyl reductase (8VR), BciA, and a nitrogenase-like enzyme, chlorophyllide a oxidoreductase (COR), respectively, to produce 3-vinyl-bacteriochlorphyllide a. Recently, we discovered 8VR activity in COR. However, the kinetic parameters of the COR 8VR activity remain unknown, while those of the COR C7=C8 reductase activity and BciA have been reported. Here, we determined the kinetic parameters of COR 8VR activity by using 8 V-Chlide. The K_m value for 8 V-Chlide was 1.4 μM, which is much lower than the 6.2 μM determined for the C7=C8 reduction of Chlide. The kinetic parameters of the dual activities of COR suggest that COR catalyzes the reduction of the C8 vinyl group of 8 V-Chlide preferentially over C7=C8 reduction when both substrates are supplied during BChl biosynthesis.     

Effect of Ionic Strength of Solution on Boron Mass Transfer by Ion Exchange Separation

Abstract

In the present study, batch kinetic tests have been performed for boron removal from model solutions using boron selective ion exchange resins Diaion CRB 02, Dowex (XUS 43594.00) and Purolite S 108. Several kinetic models have been used to evaluate the sorption kinetics of boron by means of a well mixed stirred system, diffusional models, pseudo‐first‐order, and pseudo‐second‐order kinetic models. The mass transfer model, based on a well stirred system including maximum capacity (Q_m, mg/g) and Langmuir constant (b, L/mg) values obtained from Langmuir isotherms, has been used to obtain predictive concentration changes against time. The experimental results have been used to compare with the modelling data for different ionic strength media.     

Catalytic Hydrocracking of a Bitumen‐Derived Asphaltene over NiMo/γ‐Al2O3 at Various Temperatures

Hydrocracking of a bitumen‐derived asphaltene over NiMo/γ‐Al₂O₃ was investigated in a microbatch reactor at varying temperatures. The molar kinetics of asphaltene cracking reaction was examined by fitting the experimental data. Below a defined temperature, the molar reaction showed the first‐order kinetic feature while at higher temperatures secondary reactions such as coke formation became significant, causing deviation of the reaction behavior from the proposed first‐order kinetic model. Selectivity analysis proved that dominant products varied from gases to liquids to gases with increasing temperature, shifting the dominant reaction from C–S bonds cleavage to C–C bonds cleavage.     

trans-10,cis-12 Conjugated Linoleic Acid Improved Growth Performance, Reduced Lipid Deposition and Influenced CPT I Kinetic Constants of Juvenile Synechogobius hasta

trans-10,cis-12 (t10c12) Conjugated linoleic acid (CLA) reduced body lipid deposition in various experimental animals, but the mechanisms involved were still emerging. Carnitine palmitoyltransferase I (CPT I) catalyzes an important regulatory step in lipid metabolism. At present, no studies, to our knowledge, have evaluated the kinetic constants influenced by dietary CLA in fish. In the present study, we tested the hypothesis that changes in body lipid content in fish as a response to dietary t10c12 CLA was related to the change of CPT I kinetic constants [Michaelis constant (K _m), maximal velocity and catalytic efficiency for carnitine and palmitoyl-CoA]. Juvenile Synechogobius hasta were fed three experimental diets with fish oil replaced with 0 (control), 1, or 2 % t10c12 CLA for 8 weeks. Weight gain, specific growth rate and protein efficiency rate increased with dietary t10c12 CLA level. Dietary t10c12 CLA addition significantly reduced lipid contents both in liver and muscle. Dietary CLA addition also improved CPT I activities in muscle but did not significantly influence hepatic CPT I activity. CPT I kinetic parameters (K _m, V _max and catalytic efficiency) were significantly influenced by t10c12 CLA. CPT I catalytic efficiencies with carnitine and palmitoyl-CoA as substrates were higher in muscle and liver of fish fed increasing t10c12 CLA. For the first time, the findings demonstrated effect of dietary CLA addition on CPT I kinetics in fish and supported our starting hypothesis that dietary t10c12 CLA addition induced alterations in CPT I kinetic constants of muscle and liver. Increased CPT I catalytic efficiency might be the main reason for reduced lipid deposition in these tissues by dietary t10c12 CLA supplementation.