Seasonal determinations of extracellular hydrolytic activities in heterotrophic and mixed heterotrophic/autotrophic biofilms from two contrasting rivers

The temporal changes in extracellular enzyme activities in freshwater microbial biofilms were examined in two contrasting river sites in North Wales over a 12 month period. Sites were a first order, unshaded oligotrophic upland stream (Nant Waen) and a fourth order, mildly eutrophic river with riparian tree cover (River Clywedog). When algal populations were low, biofilms of the more eutrophic site supported greater enzyme activities and higher population densities than the oligotrophic site. Composition, concentration and origin of substrates available to the respective biofilm communities influenced extracellular processing patterns. Reduction in algal populations depressed total and extracellular activities in biofilms from the first order site, suggesting that biofilm communities here were maintained by in situ primary production. Biofilms from Nant Waen were often found to contain higher extracellular activities per cell than the more eutrophic River Clywedog biofilms, which might represent the enhanced ability of an oligotrophic biofilm to accumulate extracellular enzymes. In contrast, light and darkgrown River Clywedog biofilms were not enzymatically distinct, inferring a less important role for biofilm phototrophs. Some evidence was found for increased reliance on allochthonous substrates in the River Clywedog for biofilm maintenance.     

Gluco-oligosaccharide synthesis by free and immobilized beta-glucosidase

Gluco-oligosaccharides were synthesized through the enzymatic condensation of D-glucose at high concentration using a commercial almond beta-glucosidase. The synthesis reactions were carried out with both free and immobilized enzyme, with or without sorbitol, an efficient depressor of water activity (a(w)) in the presence of different glucose concentrations. The yield and the composition of the gluco-oligosaccharides produced changed with the reaction mixture and the form of the enzyme used (free or immobilized). The use of 5 M glucose solution permitted only disaccharides to be obtained, whereas with a glucose concentration of 7.5 M glucose, di-, tri-, and tetrasaccharides were produced. A 7.5 M glucose solution used with 4.4 M sorbitol gave three times more disaccharides than the same solution without sorbitol. Moreover, the immobilized enzyme was much more active in synthesis. The synthesis yield (oligomers mg/mL . mg of enzyme) after immobilization was 573% compared to that of the free enzyme, when a 7.5 M glucose solution was tested. The effects of substrate concentration, sorbitol addition and enzyme immobilization were investigated. (c) 1993 John Wiley & Sons, Inc.     

Dramatic enhancement of enzymatic activity in organic solvents by lyoprotectants

When seven different hydrolytic enzymes (four proteases and three lipases) were lyophilized from aqueous solution containing a ligand, N-Ac-L-Phe-NH(2), their catalytic activity in anhydrous solvents was far greater (one to two orders of magnitude) than that of the enzymes lyophilized without the ligand. This ligand-induced activation was expressed regardless of whether the substrate employed in organic solvents structurally resembled the ligand. Furthermore, nonligand lyoprotectants [sorbitol, other sugars, and poly(ethylene glycol)] also dramaticaliy enhanced enzymatic activity in anhydrous solvents when present in enzyme aqueous solution prior to lyophilization. The effects of the ligand and of the lyoprotectants were nonadditive, suggesting the same mechanism of action. Excipient activated and nonactivated enzymes exhibited identical activities in water. Also, addition of the excipients directly to suspensions of nonactivated enzymes in organic solvents had no appreciable effect on catalytic activity. These observations indicate that the mechanism of the excipient-induced activation is based on the ability of the excipients to alleviate reversible denaturation of enzymes upon lyophilization. Activity enhancement induced by the excipients is displayed even after their removal by washing enzymes with anhydrous solvents. Subtilisin Carlsberg, lyophilized with sorbitol, was found to be a much more efficient practical catalyst than its "regular" counterpart. (c) 1993 John Wiley & Sons, Inc.     

Activity of mushroom polyphenol oxidase in organic medium

A kinetic study of the activity of mushroom polyphenol oxidase in an organic system was carried out to obtain detailed enzyme kinetic data in relation to optimization of reaction conditions and substrate specificity. A simple method for consistent measurement of reaction rates in the heterogeneous enzyme/organic solvent system (consisting of immobilized polyphenol oxidase and a hydrated solution of the substrate in chloroform) was designed. The aqueous content of the system was optimized using p-cresol as the substrate. With this system, a crude extract of Agaricus bisporus was used to hydroxylate and oxidize a range of selected p-substituted phenolic substrates, yielding o-quinone products. Michaelis-Menten kinetics were used to obtain apparent K(M) and V(max) values with respect to each of these substrates. Results from this analysis indicated a correlation between the enzymic kinetic parameters obtained and the steric requirements of the substrates, which could be rationalized in terms of the restricted flexibility of the enzyme when it is in chloroform and also in terms of substrate and solvent hydrophobicity. In the course of the investigation UV molar absorption coefficients of several o-quinones were measured by a novel method: (1)H nuclear magnetic resonance (NMR) spectroscopy was employed to determine component concentrations in reaction mixtures resulting from the transformation of phenols by polyphenol oxidase in chloroform. Thus the UV molar absorption coefficients could be obtained directly, avoiding the necessity to isolate the water-sensitive, unstable o-quinones. (c) 1993 John Wiley & Sons, Inc.     

Pressure affects enzyme function in organic media

Pressure affects enzyme function in nonaqueous media. Activation volumes have been determined and provide evidence that the primary effect of pressure is to enhance the stripping of water off an enzyme in polar organic solvents and leads to decreased enzymatic activity. Activation volumes of subtilisin Carlsberg in organic solvents, particularly with the enzyme hydrated, have a larger magnitude than activation volumes determined in aqueous solutions. This study provides further evidence that enzymatic activity in polar organic solvents is dominated by the interaction of enzyme-bound water with the solvent. From a practical standpoint, however, the results of this study suggest that enzymatic catalysis in organic solvents may be controlled by the combined effects of pressure and enzyme hydration. (c) 1993 John Wiley & Sons, Inc.     

Solid-state nuclear magnetic resonance investigation of solvent dependence of tyrosyl ring motion in an enzyme

Tyrosyl ring motions in alpha-lytic protease were investigated by solid-state deuterium nuclear magnetic resonance (NMR) spectroscopy in lyophilized enzyme powder, in powder suspended in organic solvents, and in aqueous crystals. Ring flipping rates were determined by examining deuterium quadrupole echo line shapes. Of the four Tyr residues in the enzyme, one was flipping at the slow (< or =10(3) s(-1)) and one at the fast (> or =10(7) s(-1)) exchange limit of the line shape experiment in all the environments tested. Flipping rates of the remaining two Tyr residues depended markedly on the solvent, with the lowest flipping rates (< or =10(3) s(-1) for both residues) observed in the enzyme powder, whether dry or suspended in hydrophobic tert-butyl methyl ether. In hydrophilic dioxane and acetonitrile, the mobility of these residues increased to 10(4) and 10(5) s(-1). The latter rate rose further to 10(6) s(-1) in the hydrated hydrophilic solvents and to > or =10(7) s(-1) in aqueous crystals. The deuterium spectrum of native alpha-lytic protease was compared with that of the enzyme whose active center was covalently modified with an inhibitor, which binds next to Tyr-123, constraining its ring. This experiment revealed that water addition to acetonitrile specifically increased the flipping rate of this active center residue. Librational motions ("wobbling"), estimated by their effect on spin-lattice relaxation times, were slowest in the anhydrous solvents, intermediate in the hydrated solvents, and fastest in the aqueous crystals. Thus, alpha-lytic protease is more rigid in organic solvents than in water, as judged by mobility of its tyrosyl residues. Water stripping by hydrophilic solvents did not increase enzyme rigidity, nor were there clear correlations between mobility and either enzymatic activity or solvent dielectric constant.     

Action of lipolytical enzymes in biphasic organic-aqueous systems: dynamics of the irreversible Michaelis-Menten reaction

Through simple model analysis, the mass action kinetic model for lipolytic enzymes in biphasic aqueous-organic systems can be simplified using the quasi-steady state assumption (or the quasi-equilibrium state assumption) for the adsorbed enzyme E* or the enzyme-substrate complex E*S. Some parameter combinations leading to the above assumptions are derived confirmed by full numerical integration of the whole enzymatic process. The results may be classified into three categories: (1) the quasi-equilibrium state assumption for E*, (2) the quasi-steady state assumption for E*, and (3) the quasi-steady state assumption for E*S. Further simplification for both E* and E*S is also discussed. (c) 1993 Wiley & Sons, Inc.     

Two bacteria causing farmer's lung: Fine structure ofThermoactinomyces vulgaris andSaccharopolyspora rectivirgula

Larval cuticle ofHelicoverpa (Heliothis)zea and yeast extract added to a minimal medium (MM) induced germination of conidia ofNomuraea rileyi whereas sterile distilled water or MM alone did not. Yeast extract increased mycelial yield, but when cuticle was added, mycelial yield significantly decreased. Proteases and chitinases ofN. rileyi were only expressed when cuticle was added to the MM.This article reports the results of research only. Mention of a proprietary product in this paper does not constitute a recommendation for use by US Department of Agriculture.     

Methyl chloride metabolism of the strictly anaerobic,methyl chloride-utilizing homoacetogen strain MC

The methyl chloride metabolism of the homoacetogenic, methyl chloride-utilizing strain MC was investigated with cell extracts and cell suspensions of the organism. Cell extracts were found to contain all enzyme activities required for the conversion of methyl chloride or of H₂ plus CO₂ to acetate. They catalyzed the dechlorination of methyl chloride with tetrahydrofolate as the methyl acceptor at a rate of 20 nmol/min × mg of cell protein. Also, the O-demethylation of vanillate with tetrahydrofolate could be measured at a rate of 40 nmol/min × mg. Different enzyme systems appeared to be responsible for the dehalogenation of CH₃Cl and for the O-demethylation of methoxylated aromatic compounds, since cells grown with methoxylated aromatic compounds exhibited a significantly lower activity of CH₃Cl conversion than methyl chloride grown cells and vice versa. In addition, ammonium thiocyanate (5 mM) completely inhibited CH₃Cl dechlorination, whereas the consumption of vanillate was not affected significantly. The data were taken to indicate, that the methyl chloride dehalogenation is catalyzed by a specific, inducible enzyme present in strain MC, and that tetrahydrofolate rather than the corrinoid-protein involved in acetate formation is the primary acceptor of the methyl group in the dechlorination reaction.     

Biological comparative study between Wolbachia-infected Aedes aegypti mosquito and Wolbachia-uninfected strain,Jeddah city,Saudi Arabia

In this study, samples of Wolbachia-infected Aedes aegypti mosquitoes were collected from Al-Safa district in Jeddah city, Saudi Arabia. The presence of Wolbachia bacteria in mosquitoes was confirmed by PCR technique and they were reared and propagated in the laboratory. Comparative studies were conducted between Wolbachia-infected A. Aegypti and the Wolbachia-uninfected laboratory strain in terms of their ability to withstand drought, resist two types of insecticides and the activities of pesticide detoxification enzymes. The Wolbachia-infected A. aegypti strain proved less able to withstand the drought period, as the egg-hatching rate of the Wolbachia-uninfected strain was greater than that of the Wolbachia-infected strain after one, two and three months of dry periods. Compared to the Wolbachia-uninfected strain, the Wolbachia-infected strain demonstrated a relatively greater resistance to tested pesticides, namely Baton 100EC and Fendure 25EC which may be attributed to the higher levels of the detoxification enzymes glutathione-S-transferase and catalase and the lower levels of esterase and acetylcholine esterase.