Characterization of a chitinase (Chit62) from Serratia marcescens B4A and its efficacy as a bioshield against plant fungal pathogens.

Chitinases have been suggested to be involved in pathogen-antagonist interaction during biological control progress of plant pathogenic fungi. Here, a recombinant bacterial chitinase originally from Serratia marcescens B4A was produced, purified, and assayed biochemically to ascertain the activity and determine the kinetics parameters. Active enzyme was used to determine its biocontrol features against fungal phytopathogens. The results demonstrated that the optimum pH and temperature for the enzyme activity were 6.0 and 55 °C, respectively. The K(m) and V(max) values were 3.30 mg ml(-1) and 0.92 units, respectively. The recombinant chitinase was demonstrated to be highly active in controlling fungal pathogens.

Detailed investigations on the solid cell culture and antimicrobial activities of the Iranian Arnebia euchroma.

In pursuit of strong shikalkin-producing cell lines, seeds of the Iranian Arnebia euchroma were collected from Dena altitudes in the central Zagross. Chemical analysis showed that the dried root of the plant contained about 8.5% (w/w) shikalkin pigment. The root explants of the young plantlets, obtained from the germinated seeds, were used for establishing callus. Then, parameters effective on proliferation and pigment production of the resulting calli were studied in detail. Accordingly, two modified media called mLS and mM9 were optimized for propagation and pigment production, respectively. Using these media, the biomass of the A. euchroma calli was increased to 600%, and the pigment production reached to a maximum of 16.3 mg per gram of the wet biomass in a period of a subculture (21 days). Parallel to these experiments, the antimicrobial activity of shikalkin pigment was examined on some fungi and gram-positive and gram-negative bacteria. Results indicated that the pigment was almost ineffective on fungi and gram-negative bacteria, but it was meaningfully effective against Micrococcus luteus.

Dual effects of aliphatic carboxylic acids on cresolase and catecholase reactions of mushroom tyrosinase.

Catecholase and cresolase activities of mushroom tyrosinase (MT) were studied in presence of some n-alkyl carboxylic acid derivatives. Catecholase activity of MT achieved its optimal activity in presence of 1.0, 1.25, 2.0, 2.2 and 3.2 mM of pyruvic acid, acrylic acid, propanoic acid, 2-oxo-butanoic acid, and 2-oxo-octanoic acid, respectively. Contrarily, the cresolase activity of MT was inhibited by all type of the above acids. Propanoic acid caused an uncompetitive mode of inhibition (K(i)=0.14 mM), however, the pyruvic, acrylic, 2-oxo-butanoic and 2-oxo-octanoic acids showed a competitive manner of inhibition with the inhibition constants (K(i)) of 0.36, 0.6, 3.6 and 4.5 mM, respectively. So, it seems that, there is a physical difference in the docking of mono- and o-diphenols to the tyrosinase active site. This difference could be an essential determinant for the course of the catalytic cycle. Monophenols are proposed to bind only the oxyform of the tyrosinase. It is likely that the binding of acids occurs through their carboxylate group with one copper ion of the binuclear site. Thus, they could completely block the cresolase reaction, by preventing monophenol binding to the enzyme. From an allosteric point of view, n-alkyl acids may be involved in activation of MT catecholase reactions.

The inhibitory effect of some new synthesized xanthates on mushroom tyrosinase activities.

Three iso-alkyldithiocarbonates (xanthates), as sodium salts, C3H7OCS2Na (I), C4H9OCS2Na (II) and C5H11OCS2Na (III), were synthesized, by the reaction between CS2 with the corresponding iso-alcohol in the presence of NaOH, and examined for inhibition of both cresolase and catecholase activities of mushroom tyrosinase (MT) from a commercial source of Agricus bisporus. 4-[(4-methylbenzo)azo]-1,2-benzendiol (MeBACat) and 4-[(4-methylphenyl)azo]-phenol (MePAPh) were used as synthetic substrates for the enzyme for the catecholase and cresolase reactions, respectively. Lineweaver-Burk plots showed different patterns of mixed and competitive inhibition for the three xanthates and also for cresolase and catecholase activities of MT. For cresolase activity, I and II showed a mixed inhibition pattern but III showed a competitive inhibition pattern. For catecholase activity, I showed mixed inhibition but II and III showed competitive inhibition. These new synthesized compounds are potent inhibitors of MT with K(i) values of 9.8, 7.2 and 6.1 microM for cresolase inhibitory activity, and also 12.9, 21.8 and 42.2 microM for catecholase inhibitory activity for I, II and III, respectively. They showed a greater inhibitory potency towards the cresolase activity of MT. Both substrate and inhibitor can be bound to the enzyme with negative cooperativity between the binding sites (alpha > 1) and this negative cooperativity increases with increasing length of the aliphatic tail in these compounds in both cresolase and catecholase activities. The cresolase inhibition is related to the chelating of the copper ions at the active site by a negative head group (S-) of the anion xanthate, which leads to similar values of K(i) for all three xanthates. Different K(i) values for catecholase inhibition are related to different interactions of the aliphatic chains of I, II and III with hydrophobic pockets in the active site of the enzyme.

The effect of some osmolytes on the activity and stability of mushroom tyrosinase.

The thermodynamical stability and remained activity of mushroom tyrosinase (MT) from Agaricus bisporus in 10 mM phosphate buffer, pH 6.8, stored at two temperatures of 4 and 40 degrees C were investigated in the presence of three different amino acids (His, Phe and Asp) and also trehalose as osmolytes, for comparing with the results obtained in the absence of any additive. Kinetics of inactivation obey the first order law. Inactivation rate constant (kinact) value is the best parameter describing effect of osmolytes on kinetic stability of the enzyme. Trehalose and His have the smallest value of kinact (0.7x10(-4) s-1) in comparison with their absence (2.5x10(-4) s-1). Moreover, to obtain effect of these four osmolytes on thermodynamical stability of the enzyme, protein denaturation by dodecyl trimethylammonium bromide (DTAB) and thermal scanning was investigated. Sigmoidal denaturation curves were analysed according to the two states model of Pace theory to find the Gibbs free energy change of denaturation process in aqueous solution at room temperature, as a very good thermodynamic criterion indicating stability of the protein. Although His, Phe and Asp induced constriction of MT tertiary structure, its secondary structure had not any change and the result was a chemical and thermal stabilization of MT. The enzyme shows a proper coincidence of thermodynamic and structural changes with the presence of trehalose. Thus, among the four osmolytes, trehalose is an exceptional protein stabilizer.

The inhibitory effect of benzenethiol on the cresolase and catecholase activities of mushroom tyrosinase.

The inhibitory effect of benzenethiol on the cresolase and catecholase activities of mushroom tyrosinase (MT) have been investigated at two temperatures of 20 and 30 degrees C in 10 mM phosphate buffer solution, pHs 5.3 and 6.8. The results show that benzenethiol can inhibit both activities of mushroom tyrosinase competitively. The inhibitory effect of benzenethiol on the cresolase activity is more than the catecholase activity of MT. The inhibition constant (K(i)) value at pH 5.3 is smaller than that at pH 6.8 for both enzyme activities. However, the K(i) value increases in cresolase activity and decreases in catecholase activity due to the increase of temperature from 20 to 30 degrees C at both pHs. Moreover, the effect of temperature on K(i) value is more at pH 6.8 for both cresolase and catecholase activities. The type of binding process is different in the two types of MT activities. The binding process for catecholase inhibition is only entropy driven, which means that the predominant interaction in the active site of the enzyme is hydrophobic, meanwhile the electrostatic interaction can be important for cresolase inhibition due to the enthalpy driven binding process. Fluorescence and circular studies also show a minor change in the tertiary structure, without any change in the secondary structure, of the enzyme due to the electrostatic interaction in cresolase inhibition by benzenethiol at acidic pH.

The inhibition effect of some n-alkyl dithiocarbamates on mushroom tyrosinase.

Three new n-alkyl dithiocarbamate compounds, as sodium salts, C4H9NHCS2Na (I), C6H13NHCS2Na (II) and C8H17NHCS2Na (III), were synthesized and examined for inhibition of both cresolase and catecholase activities of mushroom tyrosinase (MT) from a commercial source of Agaricus bisporus in 10 mM phosphate buffer pH 6.8, at 293K using UV spectrophotometry. Caffeic acid and p-coumaric acid were used as natural substrates for the enzyme for the catecholase and cresolase reactions, respectively. Lineweaver-Burk plots showed different patterns of mixed and competitive inhibition for catecholase and cresolase reactions, respectively. These new synthetic compounds can be classified as potent inhibitors of MT due to Ki values of 0.8, 1.0 and 1.8 microM for cresolase inhibitory activity, and also 9.4, 14.5 and 28.1 microM for catecholase inhibitory activity for I, II and III, respectively. They showed a greater potency in the inhibitory effect towards the cresolase activity of MT. Both substrate and inhibitor can be bound to the enzyme with negative cooperativity between the binding sites (alpha > 1) and this negative cooperativity increases with increasing length of the aliphatic tail in these compounds. The inhibition mechanism is presumably related to the chelating of the binuclear coppers at the active site and the different Ki values may be related to different interaction of the aliphatic chains of I, II and III with the hydrophobic pocket in the active site of the enzyme.

Activity and structural changes of mushroom tyrosinase induced by n-alkyl sulfates.

Catecholase activity and structural changes of mushroom tyrosinase (MT) were studied in the presence of some n-alkyl sulfate derivatives. Experiments showed that MT reached its optimal activity in the presence of 1.5, 0.6, and 0.2 mM of sodium n-octyl sulfate (SOS), sodium n-dodecyl sulfate (SDS) and sodium n-tetradecyl sulfate (STS), respectively. Native and incubated MT with the n-alkyl sulfates were also investigated from structural point of view by far-UV circular dichroism (CD) and intrinsic fluorescence spectroscopy. At the above mentioned concentrations of SOS, SDS, and STS no change in the secondary structure of MT was observed. However, changes in the tertiary structure of the enzyme due to the presence of n-alkyl sulfates were obvious. Results of this research indicate that n-alkyl sulfate with longer chain induces greater conformational changes in MT, hence, can activate it at lower concentrations.

Effects of calcium binding on the structure and stability of human growth hormone.

Thermodynamic analysis of calcium ions binding to human growth hormone (hGH) was done at 27 degrees C in NaCl solution, 50 mM, using different techniques. The binding isotherm for hGH-Ca2+ was obtained by two techniques of ionmetry, using a Ca(2+)-selective membrane electrode, and isothermal titration calorimetry. Results obtained by two ionmetric and calorimetric methods are in good agreement. There is a set of three identical and non-interacting binding sites for calcium ions. The intrinsic dissociation equilibrium constant and the molar enthalpy of binding are 52 microM and -17.4 kJ/mol, respectively. Temperature scanning UV-vis spectroscopy was applied to elucidate the effect of Ca2+ binding on the protein stability, and circular dichroism (CD) spectroscopy was used to show the structural change of hGH due to the metal ion interaction. Calcium ions binding increase the protein thermal stability by increasing of the alpha helix content as well as decreasing of both beta and random coil structures.

Stability, structural and suicide inactivation changes of mushroom tyrosinase after acetylation by N-acetylimidazole.

Modification (acetylation) of Tyr residues with N-acetylimidazole protects outstandingly mushroom tyrosinase (MT) from the suicide inactivation in the presence of its catecholic substrate, 4-[(4-methylbenzo) azo]-1,2-benzenediol. UV spectrophotometric experiments and differential scanning calorimetry (DSC) studies indicated a decrease in kinetic stability of the enzyme alongside with increase in its thermal stability as well as its stability against n-dodecyl trimethylammonium bromide as a denaturizing agent. Pace analysis resulted in standard Gibbs free energy values of 46.54 and 52.09 kJ/mol in the absence of denaturant for native and modified enzyme, respectively. Structural studies by circular dichroism (CD) spectrophotometry showed that modification did not have major impact on the secondary structure of MT; however, induced some changes in its tertiary structure. The near-UV CD results revealed that the modification had enhanced intramolecular van der Waals interactions in the enzyme structure, which was in coincidence with its thermodynamic stability.