Enzyme function in organic solvents.

Enzyme catalysis in organic solvents is being increasingly used for a variety of applications. Of special interest are the cases in which the medium is predominantly non-aqueous and contains little water. A display of enzyme activity, even in anhydrous solvents (water less than 0.02% by vol.), perhaps reflects that the minimum necessity for water is for forming bonds with polar amino acids on the enzyme surface. The rigidity of enzyme structure at such low water content results in novel substrate specificities, pH memory and the possibility of techniques such as molecular imprinting. Limited data indicates that, while enhanced thermal stability invariably results, the optimum temperature for catalysis may not change. If true in general, this enhanced thermostability would have extremely limited benefits. Medium engineering and biocatalyst engineering are relevant techniques to improve the efficiency and stability of enzymes in such low water systems. Most promising, as part of the latter, is the technique of protein engineering. Finally, this review provides illustrations of applications of such systems in the diverse areas of organic synthesis, analysis and polymer chemistry.     

Enzyme activity and flexibility at very low hydration

Recent measurements have demonstrated enzyme activity at hydrations as low as 3%. This raises the question of whether hydration-induced enzyme flexibility is important for activity. Here, to address this, picosecond dynamic neutron scattering experiments are performed on pig liver esterase powders at 0%, 3%, 12%, and 50% hydration by weight and at temperatures ranging from 120 to 300 K. At all temperatures and hydrations, significant quasielastic scattering intensity is found in the protein, indicating the presence of anharmonic, diffusive motion. As the hydration increases, a temperature-dependent dynamical transition appears and strengthens involving additional diffusive motion. The implication of these results is that, although the additional hydration-induced diffusive motion in the protein detected here may be related to increased activity, it is not required for the enzyme to function.     

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.     

Activity and flexibility of alcohol dehydrogenase in organic solvents

The oxidation of cinnamyl alcohol to cinnamaldehyde by horse liver alcohol dehydrogenase (LADH) was carried out in nearly anhydrous organic solvents and in solvents containing from 0.1 to 10% added water. In nearly anhydrous solvents containing less than 0.02% water, the oxidation rate increased as the water solubility in the solvent decreased, but the reaction did not require active LADH. Moreover, the highest activity in nearly anhydrous heptane was obtained by lyophilizing the enzyme from a solution of pH 2.0, even though LADH exhibits virtually no enzymatic activity in water at this pH. The catalytic activity of LADH was restored and increased dramatically as small amounts of water were added to each solvent. In conjunction with the activity measurements, electron paramagnetic resonance (EPR) spectroscopy and two active-site directed spin labels were used to examine solvent-dependent structural features of LADH. The EPR spectra indicated that LADH became more rigid as the dielectric constant of the solvent decreased. The degree of rigidity also depended on the pH from which the enzyme was lyophilized, indicating that the ionization state of the enzyme can have an important influence on its dynamics in organic solvents. Finally, adding 1% water to organic solvents had no apparent effect on the enzyme's conformation or flexibility near the spin label, even though enzyme activity was an order of magnitude higher when 1% water was present.     

Enzymes: An integrated view of structure, dynamics and function

Microbes utilize enzymes to perform a variety of functions. Enzymes are biocatalysts working as highly efficient machines at the molecular level. In the past, enzymes have been viewed as static entities and their function has been explained on the basis of direct structural interactions between the enzyme and the substrate. A variety of experimental and computational techniques, however, continue to reveal that proteins are dynamically active machines, with various parts exhibiting internal motions at a wide range of time-scales. Increasing evidence also indicates that these internal protein motions play a role in promoting protein function such as enzyme catalysis. Moreover, the thermodynamical fluctuations of the solvent, surrounding the protein, have an impact on internal protein motions and, therefore, on enzyme function. In this review, we describe recent biochemical and theoretical investigations of internal protein dynamics linked to enzyme catalysis. In the enzyme cyclophilin A, investigations have lead to the discovery of a network of protein vibrations promoting catalysis. Cyclophilin A catalyzes peptidyl-prolyl cis/trans isomerization in a variety of peptide and protein substrates. Recent studies of cyclophilin A are discussed in detail and other enzymes (dihydrofolate reductase and liver alcohol dehydrogenase) where similar discoveries have been reported are also briefly discussed. The detailed characterization of the discovered networks indicates that protein dynamics plays a role in rate-enhancement achieved by enzymes. An integrated view of enzyme structure, dynamics and function have wide implications in understanding allosteric and co-operative effects, as well as protein engineering of more efficient enzymes and novel drug design.     

Determination of equilibrium and individual rate constants for subtilisin-catalyzed transesterification in anhydrous environments

We report here the first determinations of individual rate constants and equilibrium constants for enzymatic reactions in essentially anhydrous organic solvents. Using the added nucleophile method we have measured the effect of changing solvent on the binding and catalytic steps for subtilisin-catalyzed transesterification of N-protected amino acid esters. The detailed information generated indicates that once the substrate has bound to the enzyme, the catalytic machinery can work at rates equivalent to those in water. The decreased overall rates for subtilisin suspended in anhydrous solvents are merely the result of extremely high values for K(s), in most cases, coupled with low concentrations of nucleophile ( approximately 1.0M in organic solvents, and 55M in water). The method described, which is generally applicable, and straightforward experimentally, will, we believe, enable a clearer understanding of how changing solvent can predictably affect the activity and specificity of the enzyme. (c) 1992 John Wiley & Sons, Inc.     

Enzyme activity below the dynamical transition at 220 K.

Enzyme activity requires the activation of anharmonic motions, such as jumps between potential energy wells. However, in general, the forms and time scales of the functionally important anharmonic dynamics coupled to motion along the reaction coordinate remain to be determined. In particular, the question arises whether the temperature-dependent dynamical transition from harmonic to anharmonic motion in proteins, which has been observed experimentally and using molecular dynamics simulation, involves the activation of motions required for enzyme function. Here we present parallel measurements of the activity and dynamics of a cryosolution of glutamate dehydrogenase as a function of temperature. The dynamical atomic fluctuations faster than approximately 100 ps were determined using neutron scattering. The results show that the enzyme remains active below the dynamical transition observed at approximately 220 K, i.e., at temperatures where no anharmonic motion is detected. Furthermore, the activity shows no significant deviation from Arrhenius behavior down to 190 K. The results indicate that the observed transition in the enzyme's dynamics is decoupled from the rate-limiting step along the reaction coordinate.     

Immobilized Candida antarctica lipase B: Hydration, stripping off and application in ring opening polyester synthesis

This work reviews the stripping off, role of water molecules in activity, and flexibility of immobilized Candida antarctica lipase B (CALB). Employment of CALB in ring opening polyester synthesis emphasizing on a polylactide is discussed in detail. Execution of enzymes in place of inorganic catalysts is the most green alternative for sustainable and environment friendly synthesis of products on an industrial scale. Robust immobilization and consequently performance of enzyme is the essential objective of enzyme application in industry. Water bound to the surface of an enzyme (contact class of water molecules) is inevitable for enzyme performance; it controls enzyme dynamics via flexibility changes and has intensive influence on enzyme activity. The value of pH during immobilization of CALB plays a critical role in fixing the active conformation of an enzyme. Comprehensive selection of support and protocol can develop a robust immobilized enzyme thus enhancing its performance. Organic solvents with a log P value higher than four are more suitable for enzymatic catalysis as these solvents tend to strip away very little of the enzyme surface bound water molecules. Alternatively ionic liquid can work as a more promising reaction media. Covalent immobilization is an exclusively reliable technique to circumvent the leaching of enzymes and to enhance stability. Activated polystyrene nanoparticles can prove to be a practical and economical support for chemical immobilization of CALB. In order to reduce the E-factor for the synthesis of biodegradable polymers; enzymatic ring opening polyester synthesis (eROPS) of cyclic monomers is a more sensible route for polyester synthesis. Synergies obtained from ionic liquids and immobilized enzyme can be much effective eROPS.     

Are water-immiscibility and apolarity of the solvent relevant to enzyme efficiency?

The question of whether the solvent's water-immiscibility is relevant to enzymatic activity was addressed by assaying four different hydrolases (three lipases and one protease) in nine anhydrous solvents of similar hydrophobicities of which four were infinitely miscible with water and five were not. For no enzyme was a jump in activity observed upon a transition from water-miscible to water-immiscible solvent. The relevance of solvent apolarity to enzymatic efficiency was also examined. To this end, three groups of isomeric anhydrous solvents were selected where within each group of isomeric anhydrous solvents were selected where within each group one solvent was apolar (i.e., lacked a permanent dipole moment). For none of the four enzymes studied was activity significantly higher in apolar solvents than in their polar counterparts. Thus we conclude that often-cited solvent's immiscibility with water and apolarity by themselves are irrelevant to enzymatic activity. (c) 1993 John Wiley & Sons, Inc.     

Characteristics of nearly dry enzymes in organic solvents: implications for biocatalysis in the absence of water

We have examined enzymes in nearly anhydrous organic solvents spanning a wide range of dielectric constants using a combination of electron paramagnetic resonance (EPR) spectroscopy, molecular dynamics simulations, high-pressure kinetic studies and the electrostatic model of Kirkwood. This approach enabled us to investigate the relationship between catalytic activity, protein flexibility and solvent polarity for an enzymatic reaction proceeding through a highly polar transition state in the near absence of water. Further insights into water-protein interactions and the involvement of water in enzyme structure and function have been obtained by EPR and multinuclear nuclear magnetic resonance studies of enzymes suspended and immobilized in organic solvents with and without added water. In these systems, correlations were observed between the water content and enzyme activity, flexibility, and active-site polarity, although the structural properties of suspended and immobilized enzymes differed markedly. These results have helped to elucidate the role of water in molecular events at the enzymic active site leading to improved biocatalysis in low-water environments.     

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.     

Hydration affects both harmonic and anharmonic nature of protein dynamics

To understand the effect of hydration on protein dynamics, inelastic neutron-scattering experiments were performed on staphylococcal nuclease samples at differing hydration levels: dehydrated, partially hydrated, and hydrated. At cryogenic temperatures, hydration affected the collective motions with energies lower than 5 meV, whereas the high-energy localized motions were independent of hydration. The prominent change was a shift of boson peak toward higher energy by hydration, suggesting a hardening of harmonic potential at local minima on the energy landscape. The 240 K transition was observed only for the hydrated protein. Significant quasielastic scattering at 300 K was observed only for the hydrated sample, indicating that the origin of the transition is the motion activated by hydration water. The neutron-scattering profile of the partially hydrated sample was quite similar to that of the hydrated sample at 100 K and 200 K, whereas it was close to the dehydrated sample at 300 K, indicating that partial hydration is sufficient to affect the harmonic nature of protein dynamics, and that there is a threshold hydration level to activate anharmonic motions. Thus, hydration water controls both harmonic and anharmonic protein dynamics by differing means.     

Hydration of enzyme in nonaqueous media is consistent with solvent dependence of its activity

Water plays an important role in enzyme structure and function in aqueous media. That role becomes even more important when one focuses on enzymes in low water media. Here we present results from molecular dynamics simulations of surfactant-solubilized subtilisin BPN' in three organic solvents (octane, tetrahydrofuran, and acetonitrile) and in pure water. Trajectories from simulations are analyzed with a focus on enzyme structure, flexibility, and the details of enzyme hydration. The overall enzyme and backbone structures, as well as individual residue flexibility, do not show significant differences between water and the three organic solvents over a timescale of several nanoseconds currently accessible to large-scale molecular dynamics simulations. The key factor that distinguishes molecular-level details in different media is the partitioning of hydration water between the enzyme and the bulk solvent. The enzyme surface and the active site region are well hydrated in aqueous medium, whereas with increasing polarity of the organic solvent (octane --> tetrahydrofuran --> acetonitrile) the hydration water is stripped from the enzyme surface. Water stripping is accompanied by the penetration of tetrahydrofuran and acetonitrile molecules into crevices on the enzyme surface and especially into the active site. More polar organic solvents (tetrahydrofuran and acetonitrile) replace mobile and weakly bound water molecules in the active site and leave primarily the tightly bound water in that region. In contrast, the lack of water stripping in octane allows efficient hydration of the active site uniformly by mobile and weakly bound water and some structural water similar to that in aqueous solution. These differences in the active site hydration are consistent with the inverse dependence of enzymatic activity on organic solvent polarity and indicate that the behavior of hydration water on the enzyme surface and in the active site is an important determinant of biological function especially in low water media.     

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.     

Preservation of Protein Dynamics in Dihydrofolate Reductase Evolution

The hydride transfer reaction catalyzed by dihydrofolate reductase (DHFR) is a model for examining how protein dynamics contribute to enzymatic function. The relationship between functional motions and enzyme evolution has attracted significant attention. Recent studies on N23PP Escherichia coli DHFR (ecDHFR) mutant, designed to resemble parts of the human enzyme, indicated a reduced single turnover rate. NMR relaxation dispersion experiments with that enzyme showed rigidification of millisecond Met-20 loop motions (Bhabha, G., Lee, J., Ekiert, D. C., Gam, J., Wilson, I. A., Dyson, H. J., Benkovic, S. J., and Wright, P. E. (2011) Science 332, 234–238). A more recent study of this mutant, however, indicated that fast motions along the reaction coordinate are actually more dispersed than for wild-type ecDHFR (WT). Furthermore, a double mutant (N23PP/G51PEKN) that better mimics the human enzyme seems to restore both the single turnover rates and narrow distribution of fast dynamics (Liu, C. T., Hanoian, P., French, T. H., Hammes-Schiffer, S., and Benkovic, S. J. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 10159–11064). Here, we measured intrinsic kinetic isotope effects for both N23PP and N23PP/G51PEKN double mutant DHFRs over a temperature range. The findings indicate that although the C-H→C transfer and dynamics along the reaction coordinate are impaired in the altered N23PP mutant, both seem to be restored in the N23PP/G51PEKN double mutant. This indicates that the evolution of G51PEKN, although remote from the Met-20 loop, alleviated the loop rigidification that would have been caused by N23PP, enabling WT-like H-tunneling. The correlation between the calculated dynamics, the nature of C-H→C transfer, and a phylogenetic analysis of DHFR sequences are consistent with evolutionary preservation of the protein dynamics to enable H-tunneling from well reorganized active sites.     

Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes

Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually neglected. The study of water dynamics in enzymes and the associated thermodynamical consequences is highly complex and has involved computer simulations, nuclear magnetic resonance (NMR) experiments, and calorimetry. Water tunnels that connect the active site with the surrounding solvent are key to solvent displacement and dynamics. The protocol herein allows for the engineering of these motifs for water transport, which affects specificity, activity and thermodynamics. By providing a biophysical framework founded on theory and experiments, the method presented herein can be used by researchers without previous expertise in computer modeling or biophysical chemistry. The method will advance our understanding of enzyme catalysis on the molecular level by measuring the enthalpic and entropic changes associated with catalysis by enzyme variants with obstructed water tunnels. The protocol can be used for the study of membrane-bound enzymes and other complex systems. This will enhance our understanding of the importance of solvent reorganization in catalysis as well as provide new catalytic strategies in protein design and engineering.     

Extraordinary enantiospecificity of lipase catalysis in organic media induced by purification and catalyst engineering

A purified lipase preparation from Candida rugosa was compared to its crude counterpart in anhydrous and slightly hydrated hydrophobic organic solvents. The purified lipase preparation was less active than the crude enzyme in dry n-heptane, whereas the presence of small concentrations of added water dramatically activated the purified enzyme but not the crude enzyme. Thus, in the presence of as little as 0.25 muL/mL of added water in n-heptane, the purified enzyme is over 230-fold more active and 6-fold more enantioselective than the dry enzyme suspension in the esterification of racemic 2-(4-chlorophenoxy)propionic acid with n-butanol. The reactivity and selectivity of this biocatalyst, however, was affected by coalescence of the enzyme preparation suspended in the wet organic solvent. Engineering the biocatalyst environment by dissolving the purified lipase in aqueous buffer and then adding this solution to n-heptane resulted in a precipitated enzyme preparation with smaller particle sizes that did not coalesce severely. In the presence of 5 muL/mL of water added with the enzyme, this pretreatment resulted in an activation over the dry, purified enzyme preparation of over 1800-fold and nearly enantiospecific catalysis (E > 100). Hence, by simply modifying the way enzymes are hydrated, dramatic activation of catalytic competency can be achieved. (c) 1996 John Wiley & Sons, Inc.     

Evolutionarily conserved linkage between enzyme fold, flexibility, and catalysis

Proteins are intrinsically flexible molecules. The role of internal motions in a protein''s designated function is widely debated. The role of protein structure in enzyme catalysis is well established, and conservation of structural features provides vital clues to their role in function. Recently, it has been proposed that the protein function may involve multiple conformations: the observed deviations are not random thermodynamic fluctuations; rather, flexibility may be closely linked to protein function, including enzyme catalysis. We hypothesize that the argument of conservation of important structural features can also be extended to identification of protein flexibility in interconnection with enzyme function. Three classes of enzymes (prolyl-peptidyl isomerase, oxidoreductase, and nuclease) that catalyze diverse chemical reactions have been examined using detailed computational modeling. For each class, the identification and characterization of the internal protein motions coupled to the chemical step in enzyme mechanisms in multiple species show identical enzyme conformational fluctuations. In addition to the active-site residues, motions of protein surface loop regions (>10 Å away) are observed to be identical across species, and networks of conserved interactions/residues connect these highly flexible surface regions to the active-site residues that make direct contact with substrates. More interestingly, examination of reaction-coupled motions in non-homologous enzyme systems (with no structural or sequence similarity) that catalyze the same biochemical reaction shows motions that induce remarkably similar changes in the enzyme–substrate interactions during catalysis. The results indicate that the reaction-coupled flexibility is a conserved aspect of the enzyme molecular architecture. Protein motions in distal areas of homologous and non-homologous enzyme systems mediate similar changes in the active-site enzyme–substrate interactions, thereby impacting the mechanism of catalyzed chemistry. These results have implications for understanding the mechanism of allostery, and for protein engineering and drug design.

Author''s Summary

Enzymes are nature''s molecular machines that catalyze biochemical reactions with remarkable efficiency. Recent evidence suggests that enzyme function may involve not only direct structural interactions between the enzyme and its substrate, but also internal motions of the enzyme itself. Here, we describe a computational investigation of three classes of enzymes that catalyze completely different biochemical reactions. Remarkably, the mobile enzyme regions and the nature of these motions are the same across species ranging from single-celled organisms to complex life-forms. Also surprisingly, non-homologous enzymes that catalyze the same chemical reaction but do not share sequence or structural similarity reveal a similar impact of enzyme motions on their reaction mechanisms. Flexible enzyme regions are found to be connected by conserved networks of coupled interactions that connect surface regions to active-site residues. These networks may provide a mechanism for the solvent on an enzyme''s surface to couple to the reaction catalyzed by the enzyme. These results have implications for understanding the mechanism of allostery (long-range effects), and for protein engineering and drug design.     

Coupling between Catalytic Loop Motions and Enzyme Global Dynamics

Catalytic loop motions facilitate substrate recognition and binding in many enzymes. While these motions appear to be highly flexible, their functional significance suggests that structure-encoded preferences may play a role in selecting particular mechanisms of motions. We performed an extensive study on a set of enzymes to assess whether the collective/global dynamics, as predicted by elastic network models (ENMs), facilitates or even defines the local motions undergone by functional loops. Our dataset includes a total of 117 crystal structures for ten enzymes of different sizes and oligomerization states. Each enzyme contains a specific functional/catalytic loop (10–21 residues long) that closes over the active site during catalysis. Principal component analysis (PCA) of the available crystal structures (including apo and ligand-bound forms) for each enzyme revealed the dominant conformational changes taking place in these loops upon substrate binding. These experimentally observed loop reconfigurations are shown to be predominantly driven by energetically favored modes of motion intrinsically accessible to the enzyme in the absence of its substrate. The analysis suggests that robust global modes cooperatively defined by the overall enzyme architecture also entail local components that assist in suitable opening/closure of the catalytic loop over the active site.     

Enzymatic catalysis in organic solvents: Polyethylene glycol modified hydrogenase retains sulfhydrogenase activity in toluene

Naturally occurring enzymes may be modified by covalently attaching hydrophobic groups that render the enzyme soluble and active in organic solvents, and have the potential to greatly expand applications of enzymatic catalysis. The reduction of elemental sulfur to hydrogen sulfide by a hydrogenase isolated from Pyrococcus furiosus has been investigated as a model system for organic biocatalysis. While the native hydrogenase catalyzed the reduction of sulfur to H(2)S in aqueous solution, no activity was observed when the aqueous solvent was replaced with anhydrous toluene. Hydrogenase modified with PEG p-nitrophenyl carbonate demonstrated its native biocatalytic ability in toluene when the reducing dye, benzyl viologen, was also present. Neither benzyl viologen nor PEG p-nitrophenyl carbonate alone demonstrated reducing capability. PEG modified cellulase and benzyl viologen were also incapable of reducing sulfur to H(2)S, indicating that the enzyme itself, and not the modification procedure, is responsible for the conversion in the nonpolar organic solvent. Sulfide production in toluene was tenfold higher than that produced in an aqueous system with equal enzyme activity, demonstrating the advantages of organic biocatalysis. Applications of bio-processing in nonaqueous media are expected to provide significant advances in the areas of fossil fuels, renewable feedstocks, organic synthesis, and environmental control technology. (c) 1996 John Wiley & Sons, Inc.