Characteristics of a mesoporous silicate immobilized trypsin bioreactor in organic media

Mesoporous silicates (MPS) materials are attractive materials for immobilizing proteins/enzymes because of their well ordered structures, large surface areas (up to 1000 m(2) g(-1)), narrow pore size distributions, large pore diameters, and pore volumes. MPS with average pore diameters ranging from 28 to 300 Angstroms were prepared using cationic and nonionic surfactants. The influence of water content, pH, storage, and thermal treatment on the activity of trypsin immobilized onto MPS was investigated. In a range of solvents, the amidolytic activity of immobilized trypsin was higher than that of the lyophilized preparation. Significant increases in k(cat)/K(M) occurred in propanol, ethanol, methanol, and formamide of 90, 62, 45 and 26, respectively. The observed increases were primarily a result of substantial increases in k(cat).     

Effects of neutral salts and alcohols on the activity of Streptomyces caespitosus neutral protease

Streptomyces caespitosus neutral protease (ScNP) is one of the smallest metalloproteinase with a molecular mass of 14 kDa. Effects of solvent composition on ScNP activity were examined using a peptide substrate. The k(cat)/K(m) values of ScNP exhibited bell-shaped pH-dependence with the optimal pH of 6.4-7.0 and the pK(a) values of 5.0 +/- 0.1 and 8.3 +/- 0.1. ScNP activity increased in an exponential fashion with increasing [NaCl]. The relative k(cat)/K(m) value at 3.6 M NaCl to that at 0 M NaCl was 3.7, and the degree of the activation at x M NaCl was expressed as 1.2 (x) (x < 2.0) and 1.4(x) (x > 2.0). On the other hand, ScNP activity decreased with increasing concentrations of LiCl, KCl, NaBr, LiBr, KBr and NaClO(4). Alcohols inhibited ScNP activity with the IC(50) values, the concentration required for decreasing the activity at 50% of the maximum, of 0.77-6.54 M. The order of the inhibitory potency was 1-butanol, 2-methyl-1-propanol, 2-methyl-2-butanol > 2-methyl-2-propanol, 2-butanol, 1-propanol > 2-propanol > ethanol > methanol. The activities recovered completely by the dilution of alcohols, suggesting that the ScNP inhibition by alcohols is reversible. These characteristics of ScNP are compared with those of human matrix metalloproteinase 7 and thermolysin.     

Combinatorial synthesis, selection, and properties of esterase peptide dendrimers

A 65,536-member combinatorial library of peptide dendrimers was prepared by split-and-mix synthesis and screened on solid support for esterolytic activity in aqueous buffer using 8-butyryloxypyrene-1,3,6-trisulfonate (2) as a fluorogenic substrate. Active sequences were identified by analysis of fluorescent beads. The corresponding dendrimers were resynthesized by solid-phase synthesis, cleaved from the resin, and purified by preparative reverse-phase HPLC. The dendrimers showed the expected catalytic activity in aqueous buffer. Catalysis was studied against a pannel of fluorogenic 8-acyloxypyrene-1,3,6-trisulfonate substrates. The catalytic peptide dendrimers display enzyme-like kinetics in aqueous buffer with substrate binding in the range K(M) approximately 0.1 mM, catalytic rate constants k(cat) approximately 0.1 min(-1), and specific rate accelerations over background up to k(cat)/k(uncat) = 10,000.     

Effects of osmolytes on hexokinase kinetics combined with macromolecular crowding: test of the osmolyte compatibility hypothesis towards crowded systems

We investigated the effect of compatible and non-compatible osmolytes in combination with macromolecular crowding on the kinetics of yeast hexokinase. This was motivated by the fact that almost all studies concerning the osmolyte effects on enzyme activity have been performed in diluted buffer systems, which are far from the physiological conditions within cells, where the cytosol contains several hundred mg protein ml(-1). Four organic (glycerol, betaine, TMAO and urea) and one inorganic (NaCl) osmolyte were tested. It was concluded that the effect of compatible osmolytes (glycerol, betaine and TMAO) on V(max) and K(M) was practically equivalent in pure buffer and in 200-250 mg BSA ml(-1) supporting the view that these small organic osmolytes do minimal perturbance on enzyme function in physiological solutions. The effect of urea on enzyme kinetics was not independent of protein concentration, since the presence of 250 mg BSA ml(-1) partly compensated the perturbing effect of urea. Even though the organic osmolytes glycerol, betaine and TMAO are generally considered compatible with enzyme function, especially glycerol did have a significant effect on hexokinase kinetics, decreasing both k(cat), K(M) and k(cat)/K(M). The osmolytes decreased k(cat)/K(M) in the order: NaCl>Urea>TMAO/glycerol>betaine. For the organic osmolytes this order correlates with the degree of exclusion from protein-water interfaces. Thus, the stronger the exclusion the weaker the perturbing effects on k(cat)/K(M).     

Activity,stability and conformational flexibility of seed coat soybean peroxidase

Seed coat soybean peroxidase (SBP) belongs to class III of the plant peroxidase superfamily that includes the classical peroxidase, namely horseradish peroxidase (HRP). We have measured the catalytic activity (k(cat)) and catalytic efficiency (k(cat)/K(M)) of SBP and that of HRP-C for the oxidation of ABTS [2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonate)] by hydrogen peroxide at 25 degrees C. We observed that the k(cat) and k(cat)/K(M) values for SBP are much higher than those for HRP-C at all pH values, rendering SBP a more potent peroxidase. This is attributed to the relatively more solvent exposed delta-meso heme edge in SBP. We observed that the maximum catalytic activity and conformational stability of SBP is at pH approximately 5.5. A pH maximum of 5.0 for the catalytic activity of SBP has recently been reported. Estimation of secondary structural elements at various pH values indicated that there is a maximal reduction of beta-strands and beta-turns at pH 5.5 causing the heme to be further exposed to the solvent and increasing the overall conformational flexibility of the protein.     

Purification and physical-chemical characterization of the three hydroperoxidases from the symbiotic bacterium Sinorhizobium meliloti

Three genes encoding heme hydroperoxidases (katA, katB, and katC) have been identified in the soil bacterium Sinorhizobium meliloti. The recombinant proteins were overexpressed in Escherichia coli and purified in order to achieve a spectral and kinetic characterization. The three proteins contain heme b with high-spin Fe(III). KatB is an acidic bifunctional homodimeric catalase-peroxidase exhibiting both catalase (k(cat) = 2400 s(-1)) and peroxidase activity and having a high affinity for hydrogen peroxide (apparent K(M) = 1.6 mM). KatA and KatC are acidic monofunctional homotetrameric catalases. Although different in size (KatA is a small subunit catalase while KatC is a large subunit catalase) both enzymes exhibit the same heme type and a similar affinity for H(2)O(2) (apparent K(M) values of 160 and 150 mM). However, the turnover rate of KatA (k(cat) = 279000 s(-1)) exceeds that of KatC (k(cat) = 3100 s(-1)) significantly. The kinetic parameters are in good agreement with the physiological role of these heme proteins. KatB is the housekeeping hydroperoxidase exhibiting the highest affinity for hydrogen peroxide, while KatA has the lowest H(2)O(2) affinity but the highest k(cat)/K(M) value (1.75 x 10(6) M(-1) s(-1)), in agreement with the hydrogen peroxide inducibility of the encoding gene. Moreover, the lower catalytic efficiency of KatC (2.1 x 10(4) M(-1) s(-1)) appears to be enough for growing in the stationary phase and/or under heat or salt stress (conditions that are known to favor katC expression).     

Influence of cosolvents on the hydrophobic surface immobilization topography of Candida antarctica lipase B

The presence of cosolvents and co-solutes during the immobilization of lipases on hydrophobic supports may influence the extent of lipase immobilization and the long-term catalytic stability of the biocatalyst. Candida antarctica B lipase immobilization was examined on a hydrophobic surface, i.e., gold modified with a methyl-terminated, self-assembled alkylthiol layer. Lipase adsorption was monitored gravimetrically using a quartz crystal microbalance (QCM). Lipase activity was determined colorimetrically by following p-nitrophenol propionate hydrolysis. Adsorbed lipase topography was examined by atomic force microscopy (AFM). Lipase adsorption from low ionic strength aqueous buffer produced a uniform confluent protein monolayer. Inclusion of 10% (vol) ethanol in the buffer during immobilization resulted in a 33% adsorbed mass increase. Chemically similar cosolvents, all at 10% by volume in buffer, were also individually examined for their influence on CALB adsorption. Glycerol or 1-propanol increased mass adsorption by 10%, while 2-propanol increased mass adsorption by 33%. QCM dissipation values increased threefold with the inclusion of either ethanol or 2-propanol in the medium during lipase adsorption, indicating formation of multilayers of CALB. Partial multilayer formation using 10% ethanol was confirmed by AFM. Inclusion of 10% ethanol in the CALB immobilization buffer decreased the specific activity of the immobilized lipase by 37%. The formation of lipase multilayers in the presence of certain cosolvents thus results in lower specific activity, which might be due to either influences on lipase conformation or substrate active site accessibility.     

Plk1 activation by Ste20-like kinase (Slk) phosphorylation and polo-box phosphopeptide binding assayed with the substrate translationally controlled tumor protein (TCTP)

The mitotic protein kinase Plk1 catalyzes events associated with centrosome maturation, kinetocore function, spindle formation, and cytokinesis and is a target for anticancer drug design. It is composed of a N-terminal kinase domain and a C-terminal polo-box domain (PBD). The PBD domain serves to localize the kinase on cognate phosphorylated substrates, and this binding relieves the inhibition of the kinase by the PBD. Similar to many protein kinases, Plk1 is activated by phosphorylation on a threonine residue, Thr210, in the activation segment. In this work, we describe expression in Escherichia coli cells and purification of full-length Plk1 in quantities suitable for structural studies and use this material for quantitative characterization of the activation events with the substrate translationally controlled tumour protein (TCTP). The presence of the PBD-binding phosphopeptide enhances phosphorylation by the activating Ste20-like kinase (Slk). Native Plk1 exhibits a basal catalytic efficiency k cat/ K(M) of 9.9 x 10 (-5) s (-1) microM (-1). Association with a polo-box-binding phosphopeptide increased the catalytic efficiency by 11x largely through an increase in k(cat) with no change in K(M). Phosphorylation by Slk increases catalytic efficiency by 202x with a 2.3-fold reduction in K(M) and 88-fold increase in k(cat). Phosphorylation and the presence of the PBD-binding phosphopeptide result in an increase in catalytic efficiency of 1515x with a 2.3-fold decrease in K(M) and a 705-fold increase in k(cat) over the unmodified Plk1. Knowledge of kinase regulatory mechanisms and the structures of the Plk1 individual domains has allowed for a model to be proposed for these activatory events.     

The self-diffusion and hydrogen bond interaction in neat liquid alkanols: a molecular dynamic simulation study

Self-diffusion of methanol, ethanol, 1-propanol and 2-propanol has been studied by molecular dynamics simulation in the temperature range between the melting pressure curve and 478 K at pressures up to 300 MPa. The simulation results on self-diffusion of methanol, ethanol and 2-propanol (for 2-propanol, at high temperatures) agree well with experiment, which suggests that the simulation method is a powerful tool to obtain self-diffusion coefficients over wide range of temperature and pressure, under which it is rather difficult for experiments. The local structures of methanol, ethanol and 2-propanol are investigated by calculating the radial distribution functions, H-bond numbers, coordination numbers and the ratios of H-bond number divided by coordination number. The correlation between self-diffusion and structural properties, and the influence of temperature and pressure on them are discussed. The degree of forming H-bond space network in methanol, ethanol and water is higher than that in 2-propanol, and they are all higher than those in ammonia and methylamine. The simulation results demonstrate that the effect of hydrogen bonding on the translational dynamics in methanol and ethanol is more pronounced than that in 2-propanol.     

Biphasic effects of alcohols on the phase transition of poly(L-lysine) between alpha-helix and beta-sheet conformations.

Poly(L-lysine) exists as a random-coil at neutral pH, an alpha-helix at alkaline pH, and a beta-sheet when the alpha-helix poly(L-lysine) is heated. The present Fourier-transform infrared (FTIR) study showed that short-chain alcohols (methanol, ethanol, and 2-propanol) partially transformed alpha-helix poly(L-lysine) to beta-sheet when their concentrations were low. At higher concentrations, however, these alcohols reversed the reaction, and the alcohol-induced beta-sheet was transformed back to alpha-helix structure. The reversal occurred at 1.40 M methanol, 0.96 M ethanol, and 0.55 M 2-propanol. The alcohol effects on the secondary structure were further investigated by circular dichroism (CD) on the thermally induced beta-sheet poly(L-lysine). Methanol, ethanol, and 1-propanol, but not 1-butanol, shifted the negative mean-residue ellipticity at 217 nm of the beta-sheet poly(L-lysine) to the positive side at low concentrations of the alcohols and to the negative side at high concentrations. With 1-butanol, only the positive-side shift was observed. The positive-side shift at low concentrations of alcohols indicates enhancement of the hydrophobic interactions among the side chains of the polypeptide in the beta-sheet conformation. The negative-side shift indicates a partial transformation to alpha-helix. The shift from the positive to negative side occurred at 7.1 M methanol, 4.6 M ethanol, and 3.1 M 1-propanol. The alcohol concentrations for the beta-to-alpha transition were higher in the CD study than in the IR study.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of non-aqueous solvents on CO2 absorption in monoethanolamine: Ab initio calculations

Monoethanolamine (MEA) is the most typical alkanolamine and its aqueous solutions are widely used for CO₂ absorption with mature technology, but the regeneration process is energy consuming. To reduce the energy demand, non-aqueous solvents, such as methanol and ethanol are proposed to substitute water in amine solutions. To understand the influence of the aqueous and non-aqueous solvents on CO₂ capture process, the chemical reactions of MEA absorbing CO₂ were conducted via ab initio calculations. The non-aqueous solvents discussed in this paper are methanol, ethanol, 1-propanol and 2-propanol. The reaction patterns were investigated and energy barriers were observed. The results show that zwitterion formation and the followed intermolecular hydrogen transfer are proven to be the most possible reaction pattern in both aqueous and non-aqueous solvents. The energy analysis shows that the forward reaction energy barriers increase while the backward barriers decrease as the solvent changes from water to methanol, ethanol, 1-propanol and 2-propanol in turn. The decreases of the energy barriers for backward processes are much higher than the corresponding increases for forward processes. These results indicate that lower energies are required in non-aqueous solvents than in water during the desorption reactions and the non-aqueous solvents are very promising to reduce the regeneration energy consumption in MEA capturing CO₂ process. Moreover, the reaction energy gaps between different solvation effects were found to have linear relationship with the logarithm of the dielectric constant difference, which could provide an easy way to theoretically predict the reaction energies of monoethanolamine absorbing CO₂ in other solvation effect and can be used to screen appropriate CO₂ capture solvent.     

Kinetics of cytochrome P450 2E1-catalyzed oxidation of ethanol to acetic acid via acetaldehyde.

The P450 2E1-catalyzed oxidation of ethanol to acetaldehyde is characterized by a kinetic deuterium isotope effect that increases K(m) with no effect on k(cat), and rate-limiting product release has been proposed to account for the lack of an isotope effect on k(cat) (Bell, L. C., and Guengerich, F. P. (1997) J. Biol. Chem. 272, 29643-29651). Acetaldehyde is also a substrate for P450 2E1 oxidation to acetic acid, and k(cat)/K(m) for this reaction is at least 1 order of magnitude greater than that for ethanol oxidation to acetaldehyde. Acetic acid accounts for 90% of the products generated from ethanol in a 10-min reaction, and the contribution of this second oxidation has been overlooked in many previous studies. The noncompetitive intermolecular kinetic hydrogen isotope effects on acetaldehyde oxidation to acetic acid ((H)(k(cat)/K(m))/(D)(k(cat)/K(m)) = 4.5, and (D)k(cat) = 1.5) are comparable with the isotope effects typically observed for ethanol oxidation to acetaldehyde, and k(cat) is similar for both reactions, suggesting a possible common catalytic mechanism. Rapid quench kinetic experiments indicate that acetic acid is formed rapidly from added acetaldehyde (approximately 450 min(-1)) with burst kinetics. Pulse-chase experiments reveal that, at a subsaturating concentration of ethanol, approximately 90% of the acetaldehyde intermediate is directly converted to acetic acid without dissociation from the enzyme active site. Competition experiments suggest that P450 2E1 binds acetic acid and acetaldehyde with relatively high K(d) values, which preclude simple tight binding as an explanation for rate-limiting product release. The existence of a rate-determining step between product formation and release is postulated. Also proposed is a conformational change in P450 2E1 occurring during the course of oxidation and the discrimination of P450 2E1 between acetaldehyde and its hydrated form, the gem-diol. This multistep P450 reaction is characterized by kinetic control of individual reaction steps and by loose binding of all ligands.     

Macrophomate synthase: characterization, sequence, and expression in Escherichia coli of the novel enzyme catalyzing unusual multistep transformation of 2-pyrones to benzoates

Macrophoma commelinae isolated from spots on leaves of Commelina communis has the ability to transform 5-acetyl-4-methoxy-6-methyl-2-pyrone (1) to 4-acetyl-3-methoxy-5-methylbenzoic acid (macrophomic acid, 2). This biotransformation includes the condensation of the 2-pyrone ring with a C3-unit precursor to form a substituted benzoic acid. We optimized conditions for induction of enzyme activity in M. commelinae, identified oxalacetate as a C3-unit precursor with cell extract, and purified the novel enzyme, macrophomate synthase. Oxalacetate inhibited the enzyme activity at a concentration higher than 5 mM, and magnesium chloride stimulated the enzyme activity. Kinetic analyses gave K(m) of 1.7 mM for 1 at 5 mM oxalacetate, K(m) of 1.2 mM for oxalacetate at 5 mM 1, and k(cat) of 0.46 s(-1) per subunit. Pyruvate was a weak substrate, with K(m) of 35.2 mM and k(cat) of 0.027 s(-1) at 5 mM 1. We cloned and sequenced a cDNA encoding the macrophomate synthase. The cDNA of 1,225 bp contained an open reading frame that encoded a polypeptide of 339 amino acid residues and 36,244 Da, the sequence of which showed no significant similarity with known proteins in a homology search with BLAST programs. Transformed E. coli cells carrying the cDNA encoding the mature protein of macrophomate synthase overproduced macrophomate synthase under the control of the T7 phage promoter induced by IPTG. The purified enzyme showed the same values of K(m) and optimum pH as the native macrophomate synthase.     

A structure-function study of a proton transport pathway in the gamma-class carbonic anhydrase from Methanosarcina thermophila

Four glutamate residues in the prototypic gamma-class carbonic anhydrase from Methanosarcina thermophila (Cam) were characterized by site-directed mutagenesis and chemical rescue studies. Alanine substitution indicated that an external loop residue, Glu 84, and an internal active site residue, Glu 62, are both important for CO(2) hydration activity. Two other external loop residues, Glu 88 and Glu 89, are less important for enzyme function. The two E84D and -H variants exhibited significant activity relative to wild-type activity in pH 7.5 MOPS buffer, suggesting that the original glutamate residue could be substituted with other ionizable residues with similar pK(a) values. The E84A, -C, -K, -Q, -S, and -Y variants exhibited large decreases in k(cat) values in pH 7.5 MOPS buffer, but only exhibited small changes in k(cat)/K(m). These same six variants were all chemically rescued by pH 7.5 imidazole buffer, with 23-46-fold increases in the apparent k(cat). These results are consistent with Glu 84 functioning as a proton shuttle residue. The E62D variant exhibited a 3-fold decrease in k(cat) and a 2-fold decrease in k(cat)/K(m) relative to those of the wild type in pH 7.5 MOPS buffer, while other substitutions (E62A, -C, -H, -Q, -T, and -Y) resulted in much larger decreases in both k(cat) and k(cat)/K(m). Imidazole did not significantly increase the k(cat) values and slightly decreased the k(cat)/K(m) values of most of the Glu 62 variants. These results indicate a primary preference for a carboxylate group at position 62, and support a proposed catalytic role for residue Glu 62 in the CO(2) hydration step, but do not definitively establish its role in the proton transport step.     

Purification and characterization of enzymes involved in the degradation of chemotactic N-formyl peptides

N-Formyl peptides are derived from proteolytic degradation/processing of bacterial and mitochondrial proteins and serve as potent chemoattractants for mammalian phagocytic leukocytes. A response to the chemotactic N-formyl peptides released by commensal bacteria in the gut region could be detrimental, leading to unwanted inflammation. Here, two enzymes that act sequentially to degrade N-formyl peptides were purified from the rat intestinal mucosal layer and biochemically characterized. The first enzyme cleaves chemotactic peptide f-MLF to release N-formylmethionine (f-Met) and dipeptide leucylphenylalanine, with a k(cat) value of 14 s(-)(1), a K(M) value of 0.60 mM, and a k(cat)/K(M) value of 22 500 M(-)(1) s(-)(1). In-gel tryptic digestion followed by mass spectral fingerprinting identified the protein as the alpha-N-acylpeptide hydrolase (or acylamino acid-releasing enzyme, EC 3.4.19.1). The second enzyme hydrolyzes N-formylmethionine into formate and methionine with a k(cat) value of 7.9 s(-)(1), a K(M) value of 3.1 mM, and a k(cat)/K(M) value of 2550 M(-)(1) s(-)(1). This protein was identified as the N-acylase IA (or N(alpha)-acyl-l-amino acid amidohydrolase, EC 3.5.1.14). Together, these two enzymes play a protective role in degrading bacterial and mitochondrial N-formylated peptides.     

Purification and characterization of Mn-peroxidase from Musa paradisiaca (banana) stem juice

Mn-peroxidase (MnP), a biotechnologically important enzyme was purified for the first time from a plant source Musa paradisiaca (banana) stem, which is an agro-waste easily available after harvest of banana fruits. MnP was earlier purified only from the fungal sources. The enzyme was purified from stem juice by ultrafiltration and anion-exchange column chromatography on diethylamino ethylcellulose with 8-fold purification and purification yield of 65%. The enzyme gave a single protein band in SDS-PAGE corresponding to molecular mass 43 kDa. The Native-PAGE of the enzyme also gave a single protein band, confirming the purity of the enzyme. The UV/VIS spectrum of the purified enzyme differed from the other heme peroxidases, as the Soret band was shifted towards lower wavelength and the enzyme had an intense absorption band around 250 nm. The K(m) values using MnSO4 and H2O2 as the substrates of the purified enzyme were 21.0 and 9.5 microM, respectively. The calculated k(cat) value of the purified enzyme using Mn(II) as the substrate in 50 mM lactate buffer (pH 4.5) at 25 degrees C was 6.7s(-1), giving a k(cat)/K(m) value of 0.32 microM(-1)s(-1). The k(cat) value for the MnP-catalyzed reaction was found to be dependent of the Mn(III) chelator molecules malonate, lactate and oxalate, indicating that the enzyme oxidized chelated Mn(II) to Mn(III). The pH and temperature optima of the enzyme were 4.5 and 25 degrees C, respectively. The enzyme in combination with H2O2 liberated bromine and iodine in presence of KBr and KI respectively. All these enzymatic characteristics were similar to those of fungal MnP. The enzyme has the potential as a green brominating and iodinating agent in combination with KBr/KI and H2O2.     

Rescue of the orphan enzyme isoguanine deaminase

Cytosine deaminase (CDA) from Escherichia coli was shown to catalyze the deamination of isoguanine (2-oxoadenine) to xanthine. Isoguanine is an oxidation product of adenine in DNA that is mutagenic to the cell. The isoguanine deaminase activity in E. coli was partially purified by ammonium sulfate fractionation, gel filtration, and anion exchange chromatography. The active protein was identified by peptide mass fingerprint analysis as cytosine deaminase. The kinetic constants for the deamination of isoguanine at pH 7.7 are as follows: k(cat) = 49 s(-1), K(m) = 72 μM, and k(cat)/K(m) = 6.7 × 10(5) M(-1) s(-1). The kinetic constants for the deamination of cytosine are as follows: k(cat) = 45 s(-1), K(m) = 302 μM, and k(cat)/K(m) = 1.5 × 10(5) M(-1) s(-1). Under these reaction conditions, isoguanine is the better substrate for cytosine deaminase. The three-dimensional structure of CDA was determined with isoguanine in the active site.     

Polyol-specific long-chain dehydrogenases/reductases of mannitol metabolism in Aspergillus fumigatus: biochemical characterization and pH studies of mannitol 2-dehydrogenase and mannitol-1-phosphate 5-dehydrogenase

Functional genomics data suggests that the metabolism of mannitol in the human pathogen Aspergillus fumigatus involves the action of two polyol-specific long-chain dehydrogenases/reductases, mannitol-1-phosphate 5-dehydrogenase (M1PDH) and mannitol 2-dehydrogenase (M2DH). The gene encoding the putative M2DH was expressed in Escherichia coli, and the purified recombinant protein was characterized biochemically. The predicted enzymatic function of a NAD(+)-dependent M2DH was confirmed. The enzyme is a monomer of 58kDa in solution and does not require metals for activity. pH profiles for M2DH and the previously isolated M1PDH were recorded in the pH range 6.0-10.0 for the oxidative and reductive direction of the reactions under conditions where substrate was limiting (k(cat)/K) or saturating (k(cat)). The pH-dependence of logk(cat) was usually different from that of log(k(cat)/K), suggesting that more than one step of the enzymatic mechanism was affected by changes in pH. The greater complexity of the pH profiles of log(k(cat)/K) for the fungal enzymes as compared to the analogous pH profiles for M2DH from Pseudomonas fluorescens may reflect sequence changes in vicinity of the conserved catalytic lysine.     

Tyrosinase: polybrene noncovalent complexes in water-ethanol mixtures

Formation of noncovalent complexes between tyrosinase from mushrooms and a cationic polyelectrolyte, polybrene (PB, poly (1,5-dimethyl-1,5-diazaundecamethyelene) bromide), was shown to activate and stabilize tyrosinase in water-ethanol mixtures. In the reaction of catechol oxidation in aqueous solutions, catalytic activity (k(cat)) of tyrosinase-PB complex ([PB]/[tyrosinase] molar ratio 100:1, per mole of polymer) in a wide range of pH was higher than that of free tyrosinase. In aqueous solutions and in water-ethanol mixtures at moderate concentrations of ethanol (10-40% v/v), the value of k(cat) of tyrosinase-PB complex exceeded the activity of free enzyme, from 1.2-2-fold, accompanied by the essential (up to 10-fold) increase in the value of the specificity constant (k(cat)/K(m)). The results are of practical importance for the construction of biocatalysts working successfully in polar organic media.     

Constitutive expression, purification and characterization of a phosphoglucomutase from Fusarium oxysporum

The phosphoglucomutase gene from a wild type Fusarium oxysporum strain (F3), was homologously expressed, under the control of the constitutive promoter of gpdA of Aspergillus nidulans. The transformant produced elevated levels of phosphoglucomutase activity compared to the wild type, a fact that facilitated the subsequent purification procedure. The enzyme (FoPGM) was purified to homogeneity applying three anion exchange and one gel filtration chromatography steps. The native enzyme revealed a monomeric structure with a molecular mass of 60 kDa, while the isoelectric point was 3.5. FoPGM was active in pH ranged from 6.0 to 8.0, with an optimum using 3-(N-morpholino)propanesulfonic acid buffer at 7.0, while loss of activity was observed when phosphate buffer was used in the above mentioned pH range. The optimal temperature for activity was 45°C but the enzyme became unstable at temperatures above 40°C. FoPGM requires the presence of a divalent cation for its function with maximum activity being obtained with Co(2+). The apparent K(m) for Co(2+) was found to be 10 μM. The enzyme was also active with other divalent metal ions such as Mn(2+), Mg(2+), Ni(2+) and Ca(2+) but to a lesser extent. The following kinetic constants were determined: v(max), 0.74 μmol mg(protein)(-1)min(-1); k(cat), 44.2 min(-1); K(m)(G1P), 0.10mM; K(m)(G1,6 diP), 1.03 μM; k(cat)/K(m)(G1P), 443 mM(-1)min(-1) and k(cat)/K(m)(G1,6 diP), 42,860 mM(-1)min(-1). The enzyme was considered to follow a Ping Pong substituted enzyme or enzyme isomerization mechanism.