Reduction of cinnamyl alcohols and cinnamaldehydes by Saccharomyces cerevisiae

The reduction of 3′,4′-dimethoxycinnamyl alcohols to phenylpropanols by Saccharomyces cerevisiae proceeds through the corresponding aldehydes. The specificity with respect to substrate structure of the two enzymatic systems involved in the above transformation (alcohol dehydrogenase and reductase) was studied. Whereas yeast alcohol dehydrogenase shows specificity for the (E) configuration of side-chain double bond, reductase does not act on 3-substituted substrates.     

On the mechanism of hydrolysis of hydantoins by d-hydantoinase from Vigna angularis: inhibition studies

Inhibition studies were performed with d-hydantoinase from Vigna angularis. These studies were based on the fact that 5,5-di-substituted hydantoins are not recognizable substrates for the enzyme. Rac-5-methyl-5-phenylhydantoin was shown to be an efficient competitive inhibitor of this d-hydantoinase (K_i=1.28 mM). (R)-5-mono-substituted hydantoins were identified as the proper substrates of the enzyme. It is proposed that this reaction is constituted by a prior fast racemization step that provides the necessary and constant feeding of (R)-5-mono-substituted isomer and a latter (R)-specific enzymatic hydrolysis. The enzyme must present a hydrophobic environment in the pro-R face and a basic residue in the pro-S face. The feedstock configuration, its molecular volume and the presence of hydrogen in position 5 of the hydantoin are the main factors responsible for the substrate specificity showed by this enzyme.     

Stereochemistry of octopine and of its isomers and their enzymatic properties

The four isomers of octopine were prepared from pyruvic acid and l- or d-arginine and from α-keto δ-guanidinovaleric acid and l- or d-alanine by reduction with sodium cyanoborohydride. The absolute configuration of d-octopine, the natural occurring isomer being S(l) at the arginine center, and R(d) at the alanine center, was confirmed enzymatically. d-Octopine is the only isomer oxidized by NAD⁺ in the presence of octopine dehydrogenase from Pecten maximus L. The isomer with configuration S(l) at the alanine center is found to be a competitive inhibitor. Isomers with R(d) configuration at the arginine center show no detectable effect on the enzymatic reaction.     

Lipase-catalyzed esterification of rutin and naringin with fatty acids of medium carbon chain

Flavonoids rutin and naringin were acylated with fatty acids of medium carbon chain (with 8–12 carbon atoms on their molecule) in a reaction catalyzed by immobilized lipase from Candida antarctica (Novozyme) in various solvent systems. The reaction parameters affecting the acylation rate and the conversion of the enzymatic process, such as the nature of the organic solvent and acyl donor used, the water activity (a_w) of the system, as well as the kinetic of the reaction have been investigated. In all cases studied, only flavonoid monoester is identified as the product, which indicates that this lipase-catalyzed esterification is regioselective. The enzymatic acylation of flavonoids seems to follow Michaelis–Menten kinetics.     

Defining a process operating window for the synthesis of 5-methyluridine by transglycosylation of guanosine and thymine

This paper describes a high yielding coupled enzymatic reaction using Bacillus halodurans purine nucleoside phosphorylase (PNP) and E. coli uridine phosphorylase (UP) for synthesis of 5-methyluridine (5-MU) by transglycosylation. Key parameters such as reaction temperature, pH, reactant loading, reactor configuration and enzyme loading were investigated. A guanosine conversion of 95% and a 5-MU yield of 85% were achieved at 1 l scale, with a productivity of 10 g l⁻¹ h⁻¹.     

Novel and Highly Efficient Regioselective Route to Helicid Esters by Lipozyme TLL

Highly regioselective acylation of helicid with fatty acid vinyl esters catalyzed by the lipase from Thermomyces lanuginosus has been successfully performed for the first time. For the enzymatic caproylation of helicid, under the optimal conditions, initial reaction rate was 33.2 mM/h, and substrate conversion and regioselectivity were greater than 99%. In addition, the acyl recognition of the enzyme in the regioselective acylation of helicid was investigated. The results showed that although 6’-O-acyl derivatives of helicid were exclusively obtained with all the tested acyl donors, the enzymatic reaction rate varied widely with different acyl donors, presumably owing to their different interactions with the active site of the lipase. It is also interesting that the different configuration of only one hydroxyl group at C-3 in helicid couldn’t affect the lipase-catalyzed esterification and helicid has the same regioselectivity as that of D-glucose and arbutin.     

In Vitro ATP Regeneration from Polyphosphate and AMP by Polyphosphate:AMP Phosphotransferase and Adenylate Kinase from Acinetobacter johnsonii 210A

In vitro enzyme-based ATP regeneration systems are important for improving yields of ATP-dependent enzymatic reactions for preparative organic synthesis and biocatalysis. Several enzymatic ATP regeneration systems have been described but have some disadvantages. We report here on the use of polyphosphate:AMP phosphotransferase (PPT) from Acinetobacter johnsonii strain 210A in an ATP regeneration system based on the use of polyphosphate (polyP) and AMP as substrates. We have examined the substrate specificity of PPT and demonstrated ATP regeneration from AMP and polyP using firefly luciferase and hexokinase as model ATP-requiring enzymes. PPT catalyzes the reaction polyP_n + AMP → ADP + polyP_n−1. The ADP can be converted to ATP by adenylate kinase (AdK). Substrate specificity with nucleoside and 2′-deoxynucleoside monophosphates was examined using partially purified PPT by measuring the formation of nucleoside diphosphates with high-pressure liquid chromatography. AMP and 2′-dAMP were efficiently phosphorylated to ADP and 2′-dADP, respectively. GMP, UMP, CMP, and IMP were not converted to the corresponding diphosphates at significant rates. Sufficient AdK and PPT activity in A. johnsonii 210A cell extract allowed demonstration of polyP-dependent ATP regeneration using a firefly luciferase-based ATP assay. Bioluminescence from the luciferase reaction, which normally decays very rapidly, was sustained in the presence of A. johnsonii 210A cell extract, MgCl₂, polyP_n=35, and AMP. Similar reaction mixtures containing strain 210A cell extract or partially purified PPT, polyP, AMP, glucose, and hexokinase formed glucose 6-phosphate. The results indicate that PPT from A. johnsonii is specific for AMP and 2′-dAMP and catalyzes a key reaction in the cell-free regeneration of ATP from AMP and polyP. The PPT/AdK system provides an alternative to existing enzymatic ATP regeneration systems in which phosphoenolpyruvate and acetylphosphate serve as phosphoryl donors and has the advantage that AMP and polyP are stabile, inexpensive substrates.     

Alkylation of transfer RNA by N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea

The alkylation of a number of purified tRNA preparations by reaction with the carcinogens, N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea was studied in order to investigate the role of nucleic acid structure on the distribution of alkylation products within the nucleotide sequence. The rate of alkylation was greatly increased by increasing the pH over the range 6 to 8 and the degree of alkylation (expressed as moles alkyl groups/mole tRNA) was directly proportional to the concentration of the nitrosamide added and independent of the amount of tRNA present. There was no significant difference in the degree of alkylation of any of the tRNA preparations tested. Reaction with N-ethyl-N-nitrosourea resulted in a degree of alkylation some 13 times less than that produced by reaction with a similar concentration of N-methyl-N-nitrosourea. The major product of the reaction was 7-alkylguanine amounting to about 80% of the total, but 3-methylcytosine, 6-O-methylguanine and 1-methyl-, 3-methyl-, and 7-methyladenine were also identified as products of the reaction of tRNA^fMet with N-methyl-N-nitrosourea.The possibility that preferential alkylation of certain residues within the polynucleotide sequence was produced by reaction with the nitrosamides was examined by degradation of the alkylated tRNA with pancreatic ribonuclease and separation of the oligonucleotide fragments by chromatography on DEAE cellulose. When tRNA^fMet which had been alkylated by reaction with N-methyl-N-nitrosourea or N-ethyl-N-nitrosourea was analysed in this way, the distribution of 7-alkylguanine was, within the limits of experimental error, in agreement with that expected for a random reaction of the alkylating agent with all of the guanosine residues throughout the molecule. A similar result was seen when tRNA^Phe was examined. These results were obtained by alkylation under conditions where the native configuration of the tRNA was maintained and show that the tertiary structure of the nucleic acid does not impart any specificity to the reaction with the nitrosamide producing 7-alkylguanine but the possibility that such specificity does exist for the minor products of alkylation cannot be excluded.     

Enzymatic hydrolysis of 1,3-1,4-beta-glucosyl oligosaccharides by 1,3-1,4-beta-glucanase from Synechocystis PCC6803: a comparison with assays using polymer and chromophoric oligosaccharide substrates

The specificity of 1,3-1,4-β-glucanase from Synechocystis PCC6803 (SsGlc) was investigated using novel substrates 1,3-1,4-β-glucosyl oligosaccharides, in which 1,3- and 1,4-linkages are located in various arrangements. After the enzymatic reaction, the reaction products were separated and determined by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). As a result, SsGlc was found to hydrolyze the pentasaccharides, which possess three contiguous 1,4-β-glycosidic linkages (cellotetraose sequence) adjacent to 1,3-β-linkage, but none of the other oligosaccharides were hydrolyzed. To further analyze the specificity, kinetic measurements were performed using polymeric substrates and 4-methylumbelliferyl derivatives of laminaribiose and cellobiose (1,3-β-(Glc)₂-MU and 1,4-β-(Glc)₂-MU). The k_cat/K_m value obtained for barley β-glucan was considerably larger than that for lichenan, indicating that SsGlc prefers 1,3-1,4-β-glucan possessing a larger amount of cellotetraose sequence. This is consistent with the data obtained for 1,3-1,4-β-glucosyl oligosaccharides. However, the k_cat/K_m value obtained for 1,4-β-(Glc)₂-MU was considerably lower than that for 1,3-β-(Glc)₂-MU, suggesting inconsistency with the data obtained from the other natural substrates. It is likely that the kinetic data obtained from such chromophoric substrates do not always reflect the true enzymatic properties.     

Enzymatic catalysis of the reversible sulfitolysis of glutathione disulfide and the biological reduction of thiosulfate esters

Rat liver supernatants were shown to contain an enzymatic activity catalyzing in both forward and reverse directions the reversible sulfitolysis of glutathione disulfide. The enzymatic sulfitolysis has maximal activity at pH 7. S-Sulfoglutathione, which is a product of the sulfitolysis, was isolated by passage through an ion-exchange column. Three different assays were applied to determine S-sulfoglutathione, viz., methods based on the ninhydrin reaction, the formation of a thiazoline derivative in strong acid, and the use of radioactively labeled glutathione. The reversal of the sulfitolysis, i.e., the reaction of S-sulfoglutathione with glutathione, was studied directly by determination of sulfite with radioactive N-ethylmaleimide, or indirectly by coupling to the NADPH- and glutathione reductase-linked reduction of glutathione disulfide.Chromatographic analysis of rat liver supernatants demonstrated that all fractions catalyzing the reversible sulfitolysis did also catalyze the previously studied thiol-disulfide interchange of glutathione and the mixed disulfide of cysteine and glutathione.The reduction of thiosulfate esters, such as S-sulfocysteine and trimethylammonium-ethylthiosulfate, with glutathione was also catalyzed by the enzyme active in the sulfitolysis, which indicates an important biosynthetic role of the enzyme in microorganisms synthesizing cysteine via S-sulfocysteine. The enzyme is also capable of participating in the formation of the naturally occurring S-sulfoglutathione.     

Perturbation Theory in the Catalytic Rate Constant of the Henri–Michaelis–Menten Enzymatic Reaction

The Henry–Michaelis–Menten (HMM) mechanism of enzymatic reaction is studied by means of perturbation theory in the reaction rate constant k ₂ of product formation. We present analytical solutions that provide the concentrations of the enzyme (E), the substrate (S), as well as those of the enzyme-substrate complex (C), and the product (P) as functions of time. For k ₂ small compared to k _?1, we properly describe the entire enzymatic activity from the beginning of the reaction up to longer times without imposing extra conditions on the initial concentrations E _o and?S _o , which can be comparable or much different.     

Potential applications of an alcohol-aldehyde/ketone oxidoreductase from thermophilic bacteria

Practical uses of a novel alcohol dehydrogenase from Thermoanaerobium brockii have been examined in crude and purified form. Stoichiometric reduction of NADP (50 mg) was demonstrated with agarose-immobilized enzyme and 0.3 (v/v) 2-propanol solution as reductant. A coenzyme recycle number of 20000 was achieved in enzymatic reactions that employed the alcohol dehydrogenase for NADPH/NADP regeneration. Gram-scale synthesis of chiral R(+) 2-pentanol was shown in a system composed of enzyme, 2-pentanone and 2-propanol as reductant. The effect of temperature, reaction time and substrate concentration on alcohol optical purity was examined. An optical purity of 80% was achieved in the enzymatic synthesis of R(+) 2-pentanol. The enzyme was easily immobilized and stable on an enzyme electrode for analytical detection of alcohols and carbonyls. T. brockii enzyme has potential applications as a commercial alcohol dehydrogenase because of broad substrate specificity and activity at high temperature or high solvent concentration, rare carbonyl si-face stereo-specificity in hydrogen transfer, and high stability and activation of immobilized enzyme.     

Effect of organic solvents on the activity and stability of halophilic alcohol dehydrogenase (ADH2) from Haloferax volcanii

The effect of various organic solvents on the catalytic activity, stability and substrate specificity of alchohol dehydrogenase from Haloferax volcanii (HvADH2) was evaluated. The HvADH2 showed remarkable stability and catalysed the reaction in aqueous?Corganic medium containing dimethyl sulfoxide (DMSO) and methanol (MeOH). Tetrahydrofuran and acetonitrile were also investigated and adversely affected the stability of the enzyme. High concentration of salt, essential to maintain the enzymatic activity and structural integrity of the halophilic enzyme under standard conditions may be partially replaced by DMSO and MeOH. The presence of organic solvents did not induce gross changes in substrate specificity. DMSO offered a protective effect for the stability of the enzyme at nonoptimal pHs such as 6 and 10. Salt and solvent effects on the HvADH2 conformation and folding were examined through fluorescence spectroscopy. The fluorescence findings were consistent with the activity and stability results and corroborated the denaturing properties of some solvents. The intrinsic tolerance of this enzyme to organic solvent makes it highly attractive to industry.     

Spectrophotometric assay for protease activity in ionic liquids using chromogenic substrates

A new spectrophotometric assay has been developed to evaluate protease activity in ionic liquids (ILs). The assay consists of two strategies to enable real-time spectrometric analysis of enzymatic reaction in ILs. First, enzymes are modified with a comb-shaped poly(ethylene glycol), PM₁₃, to obtain a transparent enzyme solution in IL. Second, a chromogenic substrate is used to follow the enzymatic reaction in IL. p-Nitroaniline-derivatized substrates are subjected to protease-catalyzed alcoholysis to release chromogenic p-nitroaniline that can be quantitatively detected by a UV-Vis spectrophotometer. By using this method, we can evaluate protease activity in ILs quite easily without separation of products from the reaction mixture. The availability of the novel assay system was demonstrated in a kinetic analysis of subtilisin-catalyzed reaction in the IL 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ([Emim][Tf₂N]) under different reaction conditions. Because two different serine proteases, subtilisin and α-chymotrypsin, substantially retained its original substrate specificity in the IL, the assay can be extended to other enzymes by using suitable chromogenic substrates.     

Kinetic mechanism and product characterization of the enzymatic peroxidation of pterostilbene as model of the detoxification process of stilbene-type phytoalexins

The enzymatic peroxidation of pterostilbene, a strong antifungal belonging to the stilbene family, by peroxidase (POX), is reported for the first time as a model of phytoalexin detoxification carried out by the enzymatic pool of pathogens. Kinetic characterization of the pterostilbene oxidation reaction pointed to an optimum pH of 7.0, at which value the thermal stability of POX was studied. Moreover, the data showed that pterostilbene inhibits POX activity at high concentrations of substrate. Several kinetic parameters, including V_max, K_m and K_I, were calculated and values of 0.16 ΔAbs min⁻¹, 14.61 μM, and 31.41 μM were reported. To understand the possible physiological role of this reaction in the phytoalexin detoxification process, the products of pterostilbene oxidation were identified using HPLC-MS and a radical-radical coupling reaction mechanism was proposed. Three main products with a high molecular weight and pronounced hydrophobicity were identified: pterostilbene cis dehydromer, pterostilbene trans dehydromer and pterostilbene open dimer. The dimeric structures of these molecules indicate that the pterostilbene oxidation reaction took place at the 4′-OH position of the hydroxystilbenic moieties and the three above mentioned dimeric products were found, due to the ability of electron-delocalized radicals to couple at various sites. Finally, the capacity of cyclodextrins (CDs) as starch model molecules in plants to complex both the substrate and the products of the oxidation reaction was evaluated. The inhibition process of POX activity was modified at high pterostilbene concentrations due to sequestering of the substrate reaction and to the different affinity of the reaction products for CDs.     

A comparison of two novel alcohol dehydrogenase enzymes (ADH1 and ADH2) from the extreme halophile Haloferax volcanii

Haloarchaeal alcohol dehydrogenases are exciting biocatalysts with potential industrial applications. In this study, two alcohol dehydrogenase enzymes from the extremely halophilic archaeon Haloferax volcanii (HvADH1 and HvADH2) were homologously expressed and subsequently purified by immobilized metal-affinity chromatography. The proteins appeared to copurify with endogenous alcohol dehydrogenases, and a double Δadh2 Δadh1 gene deletion strain was constructed to prevent this occurrence. Purified HvADH1 and HvADH2 were compared in terms of stability and enzymatic activity over a range of pH values, salt concentrations, and temperatures. Both enzymes were haloalkaliphilic and thermoactive for the oxidative reaction and catalyzed the reductive reaction at a slightly acidic pH. While the NAD⁺-dependent HvADH1 showed a preference for short-chain alcohols and was inherently unstable, HvADH2 exhibited dual cofactor specificity, accepted a broad range of substrates, and, with respect to HvADH1, was remarkably stable. Furthermore, HvADH2 exhibited tolerance to organic solvents. HvADH2 therefore displays much greater potential as an industrially useful biocatalyst than HvADH1.     

Age composition and antioxidant enzyme activities in blood of Black Sea teleosts

Age composition and age-related trends of antioxidant enzyme activities superoxide dismutase (SOD), catalase (CAT), peroxidase (PER), glutathione reductase (GR) and glutathione-S-transferase (GST) in the blood of seven Black Sea teleosts (Carangidae, Centracanthidae, Gadidae, Mullidae, Gobiidae and Scorpaenidae) collected in marine coastal area of Sevastopol (Ukraine) were studied. In the catches the animals of 1 – 2 years of age dominated while in the Scorpaena porcus population the number of relatively elder individuals belonging to classes of 3–4 years was the highest. The trends of antioxidant enzyme activities in blood were not uniform. Three types of age-dependent responses were indicated in fish blood: 1. enzymatic activity did not change with age; 2. enzymatic activity decreased with age and 3. enzyme activity increased with age or varied unclearly. The interspecies differences of age-related enzymatic activities associated with the specificity of fish biology and ecology were indicated. Despite no clear evidence of age-related differences between fish species belonging to different ecological groups both benthic forms exhibited similar age-dependent trends of SOD and PER. The correlations between blood antioxidant enzyme activities in fish belonging to suprabenthic and benthic/pelagic groups demonstrated the intermediate values as compared to the benthic and pelagic forms. The results suggest the importance of age trends for biomarkers in fish monitoring studies.     

Stereochemical studies on a plasmid-coded fluoroacetate halidohydrolase

The stereochemical course of action of haloacetate halidohydrolase H-1 from Pseudomonas sp., strain A, which catalyzes the dehalogenation of fluoroacetate to glycolate, has been determined by enzymatic analysis of products from incubations with both enantiomers of 20-fluoropropionate, and by ¹H NMR analysis of the ester of (?)-α-methoxy-α-(trifluoromethyl)phenylacetic acid with phenacyl [2-²H₁]glycolate derived from the product of incubation with the (S)-monodeuterofluoroacetate. The results support a direct displacement mechanism for this enzyme, since they indicate that the reaction is catalyzed with inversion of configuration.     

Isolation and properties of fungal β-glucosidases

Using chromatography on different matrixes, three β-glucosidases (120, 116, and 70 kDa) were isolated from enzymatic complexes of the mycelial fungi Aspergillus japonicus, Penicillium verruculosum, and Trichoderma reesei, respectively. The enzymes were identified by MALDI-TOF mass-spectrometry. Substrate specificity, kinetic parameters for hydrolysis of specific substrates, ability to catalyze the transglucosidation reaction, dependence of the enzymatic activity on pH and temperature, stability of the enzymes at different temperatures, adsorption ability on insoluble cellulose, and the influence of glucose on catalytic properties of the enzymes were investigated. According to the substrate specificity, the enzymes were shown to belong to two groups: i) β-glucosidase of A. japonicus exhibiting high specific activity to the low molecular weight substrates cellobiose and pNPG (the specific activity towards cellobiose was higher than towards pNPG) and low activity towards polysaccharide substrates (β-glucan from barley and laminarin); ii) β-glucosidases from P. verruculosum and T. reesei exhibiting relatively high activity to polysaccharide substrates and lower activity to low molecular weight substrates (activity to cellobiose was lower than to pNPG).     

Lipases are soluble and active in glycerol carbonate as a novel biosolvent

Glycerol carbonate was synthesized as biosolvent for the development of soluble enzymatic system. The effects of various reaction parameters on activity and stability of lipases were investigated using the transesterification of ethyl butyrate with n-butanol as a model reaction. Enzymatic activity in glycerol carbonate was compared with that in water and in conventional organic solvents with different ionizing and dissociating abilities. The pK_a value of trichloroacetic acid and transesterification activities of Candida antarctica lipase B and Candida rugosa lipase in glycerol carbonate are similar to those in water, indicating that ionizing and dissociating powers are capable of satisfactorily predicting the biocompatibility of organic solvents for soluble enzymatic systems.