Oxidation of lignans and lignin model compounds by laccase in aqueous solvent systems

The stability and activity of the low redox potential Melanocarpus albomyces laccase (MaL) in various aqueous organic (acetone, ethanol, propylene glycol, diethylene glycol monomethyl ether) solvent systems was studied spectrophotometrically using 2,6-dimethoxyphenol (2,6-DMP) as substrate. In addition, reactivity of the enzyme with two lignans; matairesinol (MR) and 7-hydroxymatairesinol (HMR), was examined by oxygen consumption measurements in the most potential aqueous organic solvent systems. Polymerization of the lignans by MaL was verified by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and size exclusion chromatography (SEC). Polymerization of the higher molecular weight lignin model compound, dehydrogenation polymers (DHPs), was studied by SEC. The solubilities of industrial softwood and hardwood kraft lignins were evaluated as parameters for investigation of enzymatic modification in aqueous organic solvent systems. The functioning of MaL in different aqueous organic media was excellent. Propylene glycol and diethylene glycol monomethyl ether were better solvents than ethanol or acetone in enzymatic oxidations. Even though they were the best solvents for enzyme oxidation, ethanol and propylene glycol were selected for further tests because of their different physicochemical properties. The results obtained in this study for the use of laccase-catalysed reactions in organic solvents to improve the efficiency of lignin oxidation may be exploited in several applications and areas in which the solubility of the reactants or products is a limiting factor.     

Improvement of hydrogenase enzyme activity by water-miscible organic solvents

Both stability and catalytic activity of the HynSL Thiocapsa roseopersicina hydrogenase in the presence of different water-miscible organic solvents were investigated. For all organic solvents under study the substantial raise in hydrogenase catalytic activity was observed. The stimulating effect of acetone and acetonitrile on the reaction rate rose with the increase in solvent concentration up to 80%. At certain concentrations of acetonitrile and acetone (60–80%, v/v in buffer solution) the enzyme activity was improved even 4–5 times compared to pure aqueous buffer. Other solvents (aliphatic alcohols, dimethylsulfoxide and tetrahydrofuran) improved the enzyme activity at low concentrations and caused enzyme inactivation at intermediate concentrations. The long-term incubation of the hydrogenase with aliphatic alcohols, dimethylsulfoxide and tetrahydrofuran at intermediate concentrations of the latter caused enzyme inactivation. The reduced form of hydrogenase was found to be much more sensitive to action of these organic solvents than the enzyme being in oxidized state. The hydrogenase is rather stable at high concentrations of acetone or acetonitrile during long-term storage: its residual activity after incubation in these solvents upon air within 30 days was about 50%, and immobilized enzyme remained at the 100% of its activity during this period.     

The effects of organic solvent/water mixtures on the structure and catalytic activity of porcine pepsin

The effects of aqueous solutions of ethanol, acetonitrile and 1,4-dioxane in the concentration range 10–90% (v/v) on the activity of porcine pepsin were studied. The enzyme retained its activity in aqueous ethanol and aqueous acetonitrile with increasing organic solvent concentration up to 60%, and in aqueous 1,4-dioxane up to 30%, but thereafter a considerable decrease in activity was observed. The changes caused in the catalytic activity by the water-miscible organic solvents may be related to structural changes, which were followed by means of intrinsic fluorescence and circular dichroism spectroscopy measurements.     

How do organic solvents affect peroxidase structure and function?

The effect of organic solvents on horseradish peroxidase structure and function has been studied. Some, but not complete, enzyme denaturation occurs even in low volumes of water-miscible organic solvents (e.g., greater than 30% v/v dioxane, greater than 50% v/v methanol, and greater than 20% v/v acetonitrile) as determined by the decreased difference between the fluorescence of peroxidase's sole tryptophan residue and free L-tryptophan in solution. Absorbance and electron paramagnetic resonance spectroscopies indicate exposure of peroxidase's active site to the organic solvent. This reduces the local polarity in the enzyme's active site and results in stronger hydrogen bonding of phenolic substrates to the enzyme. In extreme cases (e.g., 95% v/v dioxane, 90% v/v acetonitrile, and ethyl and butyl acetate containing 2 and 1% v/v aqueous buffer, respectively), the transition state of the enzymic reaction is sufficiently perturbed so as to alter the magnitude of the Hammett rho value. This is most likely the result of the increased strength of hydrogen bonding between electron-donating alkoxyphenols (negative sigma values) and an electrophilic group in the enzyme's active site, thereby reducing catalytic efficiencies for such substrates relative to alkyl- and chlorophenols. Perhaps the most important effect of the organic solvent, however, is the significant ground-state stabilization of phenolic substrates in organic media as opposed to aqueous buffer. This stabilization can account for nearly 4 orders of magnitude in reduction of catalytic efficiency and is manifested in increased Km's. This study indicates that enzymes can maintain much of their native active-site structure in organic media and that the effect of solvent on substrate thermodynamics must be considered.     

Preparation and catalytic performance of surfactant-manganese peroxidase-Mn(II) ternary complex in organic media

A novel preparation method for surfactant-MnP-Mn(II) ternary complex utilizing water-in-oil emulsions has been developed. The surfactant-MnP complex was spectroscopically characterized, strongly suggesting that the heme environment of the surfactant-MnP complex in benzene is identical to that of native MnP in the aqueous buffer. o-Phenylenediamine oxidation catalyzed by the surfactant-MnP-Mn(II) ternary complex was performed in benzene. The ternary complex efficiently catalyzed the oxidation, and the complex was catalytically stable. Kinetic experiments revealed that the reaction mechanism was as follows: MnP is oxidized by H(2)O(2) and the oxidized intermediate catalyzes the oxidation of Mn(II) to Mn(III) and the latter, after complexed with malonate, readily oxidizes o-PDA inside the complex. Thus, the organic substrate o-PDA, but not Mn(III), shuttled between the surfactant-MnP-Mn(II) ternary complex and organic solvent.     

Bioremediation of polycyclic aromatic hydrocarbons by fungal laccases engineered by directed evolution

Fungal laccases are useful for several remarkable transformations, such as bioremediation of polycyclic aromatic hydrocarbons (PAHs), synthesis of phenolic-based resins, oxidation of lignin derivatives and others. Most of these substrates are barely water-soluble, and although polar organic co-solvents may be added to enhance their solubility, transformation rates dramatically decrease due to the negative effect of organic solvents on the protein structure. Laccase from Myceliophthora thermophila variant T2 (MtLT2) has been submitted to laboratory evolution in Saccharomyces cerevisiae with the aim of improving activity and stability in organic co-solvents. Some 4500 clones created by random mutagenesis were screened in two rounds of directed evolution. Libraries were explored under increasing concentrations of acetonitrile and ethanol, and several mutants with improved features were purified and further characterised. Turnover rates of MtLT2 in 30% (v/v) acetonitrile and 50% (v/v) ethanol were increased up to 6.5- and 7.5-fold, respectively. The best variants showed similar rates in 20% (v/v) acetonitrile or 30% (v/v) ethanol as the parent type in aqueous media. Mutant laccases were also tested for the oxidation of anthracene in the presence of 20% (v/v) acetonitrile.     

Bioremediation of polycyclic aromatic hydrocarbons by fungal laccases engineered by directed evolution

Fungal laccases are useful for several remarkable transformations, such as bioremediation of polycyclic aromatic hydrocarbons (PAHs), synthesis of phenolic-based resins, oxidation of lignin derivatives and others. Most of these substrates are barely water-soluble, and although polar organic co-solvents may be added to enhance their solubility, transformation rates dramatically decrease due to the negative effect of organic solvents on the protein structure. Laccase from Myceliophthora thermophila variant T2 (MtLT2) has been submitted to laboratory evolution in Saccharomyces cerevisiae with the aim of improving activity and stability in organic co-solvents. Some 4500 clones created by random mutagenesis were screened in two rounds of directed evolution. Libraries were explored under increasing concentrations of acetonitrile and ethanol, and several mutants with improved features were purified and further characterised. Turnover rates of MtLT2 in 30% (v/v) acetonitrile and 50% (v/v) ethanol were increased up to 6.5- and 7.5-fold, respectively. The best variants showed similar rates in 20% (v/v) acetonitrile or 30% (v/v) ethanol as the parent type in aqueous media. Mutant laccases were also tested for the oxidation of anthracene in the presence of 20% (v/v) acetonitrile.     

Papain in organic solvents: determination of conditions suitable for biocatalysis and the effect on substrate specificity and inhibition

Synthesis of N-CBZ-(N-Carbobenzoxy)-1-amino-acid methyl esters from N-CBZ-amino acids and methanol has been used as an assay to examine the properties of papain in organic solvents containing small amounts of water. Papain is active in solvents ranging in polarity from acetonitrile to tetrachloromethane. The optimal activity in each solvent varied only about three to four fold, but the amount of added water required to achieve it varied from 4% (v/v) in acetonitrile to 0.05% (v/v) in tetrachloromethane. The enzyme was generally more stable in hydrophobic solvents and at lower water contents. The apparent K(m) value of CBZ-glycine was 26 times higher in acetonitrile than in toluene due to differential partitioning of the substrate between aqueous and organic phases. The substrate specificity of the enzyme was qualitatively little different from that in aqueous solution, with amino acid derivatives still the best substrates. Nitrile analogs of substrates inhibited the enzyme, as they do in aqueous solution, and inhibition by a variety of substituted aromatic hydrocarbons showed that the main specificity of papain for hydrophobic side chains at its S(2) subsite, was little affected. The results show that papain can catalyze reactions under a variety of conditions in organic solvents but its substrate specificity is little changed from that in aqueous media.     

Stability of hydrolytic enzymes in water-organic solvent systems

The effects of organic solvents on the stabilities of bovine pancreas trypsin, chymotrypsin, carboxypeptidase A and porcine pancreas lipase were studied. Water-miscible solvents (ethanol, acetonitrile, 1,4-dioxane and dimethyl sulfoxide) and water-immiscible solvents (ethyl acetate and toluene) were used in 100 mM phosphate buffer (pH 7.0) or 100 mM Tris/HCl buffer (pH 7.0) in concentrations of 20–80% (v/v). All hydrolytic enzymes studied were inactivated by mixtures containing dimethyl sulfoxide at higher concentrations. Trypsin and carboxypeptidase A resisted solvent mixtures containing acetonitrile, 1,4-dioxane and ethanol. They preserved more than 80% of their starting activities during 20-min incubations. The activities of lipase and chymotrypsin decreased with increasing concentration of water-miscible polar organic solvents, but at higher concentrations (80%) 70–90% of the activity remained. In mixtures with water-immiscible solvents, the decrease in activity of carboxypeptidase A was pronounced. Trypsin and chymotrypsin underwent practically no loss in activity in the presence of toluene or ethyl acetate. In respect of stability, the polar solvent proved to be more favorable for lipase. These results suggest that the conformational stabilities of hydrolytic enzymes are highly dependent on the solvent-protein interactions and the enzyme structure.     

Quantitative correlations relating the interaction of Zn(II)-tetraphenylporphine and horseradish peroxidase with amines

Reactions of nucleophilic substitution and enzymatic processes involving metalloporphirins (MP) are considered in terms of coordination of zinc(II)tetraphenylporphine (Zn-TPhP) with corresponding ligands/nucleophiles/substrates/bases. Linear correlations are performed between kinetic parameters of the Zn-TPhP coordination processes in chloroform (stability constants) and reactions of nucleophilic substitution both in aqueous and organic solvents involving pyridines, pyridine N-oxides, anilines, primary amines, as well as in reactions of oxidation of anilines with horseradish peroxidase in aqueous media (rate constants). Thermodynamic parameters of the complex formation and nucleophilic substitution linearly correlate with each other in the case of pyridines, anilines, and primary amines.     

Polymerization of guaiacol by lignin-degrading manganese peroxidase from Bjerkandera adusta in aqueous organic solvents

 Lignin-degrading manganese (II) peroxidase (MnP) purified from the culture of a wood-rotting basidiomycete, Bjerkandera adusta, was used in the polymerization of guaiacol. MnP was found to catalyze polymerization of guaiacol in 50% aqueous acetone, dimethyl formamide, methanol, ethanol, dioxane, acetonitrile, ethylene glycol and methylcellosolve. Maximum yield of polyguaiacol was achieved in 50% aqueous acetone. The weight average molecular weight (M _w) of the polymer was estimated to be 30 300 by gel permeation chromatography. However, matrix-assisted laser desorption ionization time of flight mass spectroscopy (MALDI-TOF-MS) analysis gave a more reliable M _w of 1690. IR, ¹³C-NMR, MALDI-TOF-MS and pyrolysis GC-MS analyses showed the presence of C–C and C–O linkages and quinone structure in polyguaiacol. It was also indicated that polyguaiacol has a methoxy-phenyl group as the terminal moiety. This suggests that polyguaiacol is a branched polymer in which guaiacol units are cross-linked at the phenolic group. Thermal gravimetric and differential scanning calorimetric analyses were also carried out. MnP also catalyzed the polymerization of o-cresol, 2,6-dimethoxyphenol and other phenolic compounds and aromatic amines. M _w of these polymers ranged from around 1000 to 1500. Received: 2 August 1999 / Received revision: 10 December 1999 / Accepted: 4 January 2000     

Correlation of inhibition of thermolysin by water-miscible alcoholic solvents with their physicochemical parameters and the status of monoalcoholic character of water in the peptide synthesis of Z-Phe-Phe-OMe in water organic one-phase reaction system

Inhibition of thermolysin-catalyzed peptide synthesis of N-(benzyloxycarbonyl)-L-phenylalanyl-L-phenylalanine methylester (Z-Phe-Phe-OMe) in aqueous organic one-phase reaction system by water-miscible alcoholic organic solvents correlated with most of their physicochemical parameters. Structurally similar monoalcohols and diols, including water, showed a linear relationship between inhibition and physicochemical parameters. The structure of organic solvents thus plays the key role in determining enzymatic activity in reaction media containing organic solvents.     

Synthesis of levan in water-miscible organic solvents

The synthesis of levan using a levansucrase from a strain of Bacillus subtilis was studied in the presence of the water-miscible solvents: acetone, acetonitrile and 2-methyl-2-propanol (2M2P). It was found that while the enzyme activity is only slightly affected by acetone and acetonitrile, 2M2P has an activating effect increasing the total activity 35% in 40-50% (v/v) 2M2P solutions at 30 degrees C. The enzyme is highly stable in water at 30 degrees C; however, incubation in the presence of 15 and 50% (v/v) 2M2P reduced the half-life time to 23.6 and 1.8 days, respectively. This effect is reversed in 83% 2M2P, where a half-life time of 11.8 days is observed. The presence of 2M2P in the system increases the transfer/hydrolysis ratio of levansucrase. As the reaction proceeds with 10% (w/v) sucrose in 50/50 water/2M2P sucrose is converted to levan and an aqueous two-phase system (2M2P/Levan) is formed and more sucrose can be added in a fed batch mode. It is shown that high molecular weight levan is obtained as an hydrogel and may be easily recovered from the reaction medium. However, when high initial sucrose concentrations (40% (w/v) in 50/50 water/2M2P) are used, an aqueous two-phase system (2M2P/sucrose) is induce, where the synthesized levan has a similar molecular weight distribution as in water and remains in solution.     

Comparative evaluation of manganese peroxidase- and Mn(III)-initiated peroxidation of C18 unsaturated fatty acids by different methods

The peroxidation of C18 unsaturated fatty acids by fungal manganese peroxidase (MnP)/Mn(II) and by chelated Mn(III) was studied with application of three different methods: by monitoring oxygen consumption, by measuring conjugated dienes and by thiobarbituric acid-reactive substances (TBARS) formation. All tested polyunsaturated fatty acids (PUFAs) were oxidized by MnP in the presence of Mn(II) ions but the rate of their oxidation was not directly related to degree of their unsaturation. As it has been shown by monitoring oxygen consumption and conjugated dienes formation the linoleic acid was the most easily oxidizable fatty acid for MnP/Mn(II) and chelated Mn(III). However, when the lipid peroxidation (LPO) activity was monitored by TBARS formation the linolenic acid gave the highest results. High accumulation of TBARS was also recorded during peroxidation of linoleic acid initiated by MnP/Mn(II). Action of Mn(III)-tartrate on the PUFAs mimics action of MnP in the presence of Mn(II) indicating that Mn(III) ions are involved in LPO initiation. Although in our experiments Mn(III) tartrate gave faster than MnP/Mn(II) initial oxidation of the unsaturated fatty acids with consumption of O₂ and formation of conjugated dienes the process was not productive and did not support further development of LPO. The higher effectiveness of MnP/Mn(II)-initiated LPO system depends on the turnover of manganese provided by MnP. It is proposed that the oxygen consumption assay is the best express method for evaluation of MnP- and Mn(III)-initiated peroxidation of C18 unsaturated fatty acids.     

Treatment of Candida Rugosa Lipase with Short-Chain Polar Organic Solvents Enhances its Hydrolytic and Synthetic Activities

Following a simple and quick treatment based on dissolving the crude lipase from Candida rugosa in different percentages (v/v) of several polar organic solvents (methanol, ethanol, 1 and 2-propanol, 1 and 2-butanol and acetone) followed by dialysis, different preparations with enhanced activities were obtained. The opening of the lid covering the active site is proposed as the reason for explaining the activity enhancement, both in aqueous and anhydrous organic media.     

Manganese Peroxidase-Dependent Oxidation of Glyoxylic and Oxalic Acids Synthesized by Ceriporiopsis subvermispora Produces Extracellular Hydrogen Peroxide

The ligninolytic system of the basidiomycete Ceriporiopsis subvermispora is composed of manganese peroxidase (MnP) and laccase. In this work, the source of extracellular hydrogen peroxide required for MnP activity was investigated. Our attention was focused on the possibility that hydrogen peroxide might be generated by MnP itself through the oxidation of organic acids secreted by the fungus. Both oxalate and glyoxylate were found in the extracellular fluid of C. subvermispora cultures grown in chemically defined media, where MnP is also secreted. The in vivo oxidation of oxalate was measured; ¹⁴CO₂ evolution was monitored after addition of exogenous [¹⁴C]oxalate to cultures at constant specific activity. In standard cultures, evolution of CO₂ from oxalate was maximal at day 6, although the MnP titers were highest at day 12, the oxalate concentration was maximal (2.5 mM) at day 10, and the glyoxylate concentration was maximal (0.24 mM) at day 5. However, in cultures containing low nitrogen levels, in which the pH is more stable, a better correlation between MnP titers and mineralization of oxalate was observed. Both MnP activity and oxidation of [¹⁴C]oxalate were negligible in cultures lacking Mn(II). In vitro assays confirmed that Mn(II)-dependent oxidation of [¹⁴C]oxalate by MnP occurs and that this reaction is stimulated by glyoxylate at the concentrations found in cultures. In addition, both organic acids supported phenol red oxidation by MnP without added hydrogen peroxide, and glyoxylate was more reactive than oxalate in this reaction. Based on these results, a model is proposed for the extracellular production of hydrogen peroxide by C. subvermispora.     

Effect of water-miscible aprotic solvents on kyotorphin synthesis catalyzed by immobilized α-chymotrypsin

Summary Immobilized -chymotrypsin was used as catalyst to synthesize a kyotorphin derivative (Bz-Tyr-Arg-OEt) in the presence of five water-miscible aprotic solvents (dimethylsulphoxide, dimethylformamide, acetonitrile, acetone and tetrahydrofurane) at 30 °C. By using a kinetically-controlled approach, the maximum synthetic activity was obtained when Arg-OEt was used as nucleophile donor at a concentration 1.5-times higher than the acyl-acceptor substrate (Bz-Tyr-OEt). The water-miscible aprotic solvents enhanced greatly the synthetic activity proportionally to their hidrophilicity properties adequately measured by the log P parameter. At the optimum solvent concentration for the enzymatic peptide synthesis, both the water activity (Aw) of the media and the water content of the immobilized derivative showed a saturation profile against the log P parameter. As a function of the solvent hydrophilicity, these water parameters were shown as key parameters for the increase in the synthetic activity of the enzyme by the presence of these solvents.     

Glucose condensation by glucoamylase in organic solvents

Summary Oligosaccharides were synthesized through the enzymatic condensation of D-glucose by glucoamylase in water-organic mixtures with high concentrations of two of diethylene glycol diethyl ether or triethylene glycol dimethyl ether. The effect of water content on the yield of reaction was studied; maximum yield was obtained with 10% (v/v) of water in the two systems. Kinetics of synthesis and products composition were different with the two solvents. 37% of glucose were condensed by action of glucoamylase from a reaction medium containing 20 g/L of glucose and 90% (v/v) of diethylene glycol diethyl ether.     

Influence of Water Miscible Organic Solvents on α-chymotrypsin in Solution and Immobilized on Eupergit CM

The effects of the water-miscible organic solvents (methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, N,N′-dimethylformamide and tetrahydrofuran) on the stability and catalytic activity of α-chymotrypsin (CT) immobilized on Eupergit CM were studied. Enhanced stabilities and activities were observed both as a consequence of immobilization and the presence of organic solvent, which in combination provide long term (at least 24 h) retention of activity, and up to 50-fold increase in 50% (v/v) methanol in buffer. Low quantities (20%, v/v) of acetonitrile not only prevented CT inactivation by autolysis at 20°C but also induced a significant increase in the activity of both free (six-fold) and immobilized (two-fold) CT.Linus Olofsson and Pernilla Söderberg authors have contributed equally to the work.     

Correlation between the physicochemical properties of organic solvents and their biocompatibility toward epoxide hydrolase activity in whole-cells of a yeast, Rhodotorula sp.

Epoxides are often highly hydrophobic substrates and the presence of an organic co-solvent within an aqueous bioreactor is in such cases indicated. The effect of 40 water-miscible and -immiscible organic solvents on epoxide hydrolase activity in whole-cells of the yeast Rhodotorula sp. UOFS Y-0448 was investigated. No formal correlation between solvent biocompatibility and physicochemical properties was deductible, although the introduction of hydroxyl groups increased biocompatibility. 1-Pentanol, 2-methylcyclohexanol and 1-octanol were the most biocompatible resulting in relatively low activity losses when used at up to 20% (v/v).