Effect Of Water-miscible Solvents Research Articles

Hypoxanthine-Guanine Phosphoribosyltransferase/adenylate Kinase From Zobellia galactanivorans: A Bifunctional Catalyst for the Synthesis of Nucleoside-5′-Mono-, Di- and Triphosphates

In our search for novel biocatalysts for the synthesis of nucleic acid derivatives, we found a good candidate in a putative dual-domain hypoxanthine-guanine phosphoribosyltransferase (HGPRT)/adenylate kinase (AMPK) from Zobellia galactanivorans (ZgHGPRT/AMPK). In this respect, we report for the first time the recombinant expression, production, and characterization of a bifunctional HGPRT/AMPK. Biochemical characterization of the recombinant protein indicates that the enzyme is a homodimer, with high activity in the pH range 6-7 and in a temperature interval from 30 to 80°C. Thermal denaturation experiments revealed that ZgHGPRT/AMPK exhibits an apparent unfolding temperature (Tm) of 45°C and a retained activity of around 80% when incubated at 40°C for 240 min. This bifunctional enzyme shows a dependence on divalent cations, with a remarkable preference for Mg2+ and Co2+ as cofactors. More interestingly, substrate specificity studies revealed ZgHGPRT/AMPK as a bifunctional enzyme, which acts as phosphoribosyltransferase or adenylate kinase depending upon the nature of the substrate. Finally, to assess the potential of ZgHGPRT/AMPK as biocatalyst for the synthesis of nucleoside-5′-mono, di- and triphosphates, the kinetic analysis of both activities (phosphoribosyltransferase and adenylate kinase) and the effect of water-miscible solvents on enzyme activity were studied.

Effects of water-miscible solvents and polyhydroxy compounds on the structure and enzymatic activity of thermolysin

The effect of organic solvents (n-propanol, isopropanol, dimethylformamide and dimethylsulfoxide) on the structure, activity and stability of thermolysin was the focus of this investigation. Results show the ability of the solvents to cause mixed inhibition of thermolysin, which was indicated by kinetic and structural studies (near-UV CD spectra and intrinsic fluorescence). Inhibitory effect of the solvents increased with increments in solvents logP. Thermoinactivation of thermolysin was studied at 80 degrees C in 50% of solvents and showed that with the increase in solvent hydrophobicity, thermal stability of the enzyme decreased. For the stabilization of thermolysin at high temperature, additives such as glycerol, sorbitol and trehalose were employed. In the presence of DMF with a relatively low logP, trehalose was shown to be a good stabilizer, whereas glycerol had a marked stabilization effect in the presence of n-propanol and isopropanol with a relatively high logP. Consequently, it was concluded that the stabilizing effect of additives can be correlated with the logP of solvents.

Substrate specificity and effects of water-miscible solvents on the activity and stability of extracellular lipase from Streptomyces rimosus

Substrate specificity, regioselectivity and transesterification activity of purified extracellular lipase from Streptomyces rimosus were investigated. The enzyme showed pronounced lipolytic activity toward a number of triacylglycerols and oils of vegetable and animal origin. It hydrolyzed most efficiently medium chain length fatty acid glycerol esters (C8–C12). There was preference for the esters of C16 and C18 unsaturated fatty acids over C16 and C18 saturated fatty acid esters, as well as for triacylglycerol substrate with cis double bond (triolein) versus trans double bond (trielaidin). Streptomyces rimosus lipase hydrolyzed primary and secondary ester bonds in triacylglycerols (triolein and 2,3-dimercapto-1-propanol tributyrate). The lipase catalyzed the hydrolysis of poly(oxyethylene) sorbitan monoesters (Tween 20–80) with rate comparable for that determined with triacylglycerols and oils. Several water-miscible solvents enhanced the lipase activity. 1,4-Dioxane activated the enzyme in a broad concentration range, up to 4-fold. Lipase was stable in solvent mixtures containing 50% (v/v) ethanol, 1,4-dioxane, acetonitrile or acetone. Tetrahydrofuran and N,N-dimethylformamide (both 50%) inactivated the enzyme with t 1/2 of 5 min and t 1/2 of 2 h, respectively. Transesterification of racemic 1-phenyl ethanol with vinyl acetate, catalyzed by extracellular lipase from Streptomyces rimosus in n-hexane, proceeded with partial R-enantioselectivity.

Effect of Water Miscible Organic Solvents on Kinetics of a Thermostable β-Glycosidase

A thermostable β-D-glycosidase from Sulfolobus solfataricus was used to determine the effect of water-miscible solvents on enzyme activity and kinetics. Various o- and p-nitrophenyl glycosides were used as representative substrates for hydrolysis. Initial enzyme activity was enhanced in the presence of methyl acetate, butan-2-one and acetonitrile. The activation by solvent was found to be transitory. 1,4-Dioxane, formamide and tetrahydrofuran were inhibitory at all concentrations with all substrates used. Kinetic studies in the presence of solvents suggested that KM values for all substrates were unaffected by methyl acetate, but increased or decreased in the presence of 1,4-dioxane or tetrahydrofuran, in a substrate dependent manner. Studies using an competitive inhibitor supported these results. Methyl acetate did not change competitive-type inhibition by D-gluconic acid lactone, while in the presence of 1,4-dioxane altered the inhibition kinetics to mixed-type. Tetrahydrofuran affected the mode of inhibition but not in a identifiable manner. These results suggest that both 1,4-dioxane and tetrahydrofuran may have a direct influence on the active site of the catalyst.

Cosolvent effect on Δ1-steroid-reductase activity of free and PAAH entrapped Mycobacterium Sp. NRRL B-3805 cells

The effect of water miscible solvents on Δ1-steroid reduction by free and polyacrylamide-hydrazide (PAAH) entrapped Mycobacterium sp. NRRL B-3805 cells was investigated. On the basis of retention of reductase activity an order of preference of diols (e.g. ethyleneglycol) > DMSO > DMF and monoalcohols (e.g. ethanol) as cosolvents was recorded. Significant increase in substrate (1,4-androstadiene-3,17-dione) solubility was attained in presence of the cosolvent of choice (ethyleneglycol), accompanied by some inhibition of the Δ1-reductase activity. Optimization of ethyleneglycol concentration (10–20% (v/v)) led to specific activity in a homogeneous medium, higher than recorded in the absence of cosolvent. Immobilization in PAAH gel resulted in high retention of immobilized enzymic activity, accompanied by minor diffusional limitations. Moreover, the gel exhibited protective effect of the entrapped cells from cosolvent inhibition. Modification of gel composition (e. g. hydrophobicity) had no significant effect on reaction kinetics.

The effect of water-miscible solvents on the ?1-dehydrogenase activity of free and PAAH-entrapped Arthrobacter simplex

The effect of water-miscible cosolvents on biotransformations of poorly water-soluble substrates by immobilized cells was investigated, using Δ1-dehydrogenation of hydrocortisone by Arthrobacter simplex as a model. Criteria for solvent selection on the basis of retention of enzymic activity were postulated and tested. Diols were considered to be the most suitable group of solvents. Substrate solubility increased tenfold in 30% (v/v) ethylene glycol, but reaction rates were significantly slower in such solutions. This was mainly caused by a decrease of oxygen solubility in the presence of the cosolvent and conformational changes imposed on the intracellular enzyme by cosolvent molecules penetrating the cell. The inhibition could be eliminated by the addition of an artificial electron acceptor, phenazine methosulphate (PMS). Reaction rates faster than those for substrate suspensions (no cosolvent added) could thus be achieved. Immobilization of Arthrobacter simplex in cross-linked polyacrylamide hydrazide gave high retentions of activity. PMS exhibited toxic effects on the entrapped cells, leading to reduced activity after extended use.

The color reactions of sulfonazo-III isomers with lanthanum and alkaline earth metal ions

Six sulfonazo-III isomers were synthesized, and their color reactions with alkaline earth metals and lanthanum investigated. All six reagents had good sensitivities for lanthanum (ϵ = 1.9–5.0·10 4), but the sensitivities for alkaline earth metals were fairly low except for the barium chelate of sulfonazo-III (ϵ = 2.8 · 10 4). The position of the sulfo group on the phenyl ring and the symmetry of the reagent are important for the barium reactions, but of little significance for the lanthanum reactions. The effects of water-miscible solvents are also described.