Spectroscopic investigation of lipase from Pseudomonas cepacia solubilized in 1,4‐dioxane by non‐covalent complexation with methoxypoly(ethylene glycol)

Lipase from Pseudomonas cepacia was made soluble in 1,4‐dioxane by lyophilization of the enzyme from aqueous solutions containing methoxypoly(ethylene glycol) (PEG). The solubility of the enzyme–PEG complex depended both on protein concentration and PEG protein ratio. Intrinsic protein fluorescence and far‐ and near‐UV circular dichroism revealed that not only did the enzyme not unfold in the organic solvent, but rather became more compact. This was seen by the slight quenching of fluorescence intensity and by the enhancement of the near‐UV circular dichroism negative signals, which are indicative of stronger interactions of tryptophanyl and/or tyrosyl residues among themselves or with other parts of the enzyme molecule. The specific activity of the lipase–PEG complex in the organic solvent was at least 2 orders of magnitude higher than that of the enzyme powder. This can be attributed both to the maintenance of native conformation and to enzyme dissolution in the reaction medium which should minimize possible limitations to enzyme–substrate interactions. © 1999 John Wiley & Sons, Inc., Biotechnol Bioeng 64: 624–629, 1999.     

Water plays a different role on activation thermodynamic parameters of alcoholysis reaction catalyzed by lipase in gaseous and organic media

The effect of water on the alcoholysis of methyl propionate and n-propanol catalyzed by immobilized Candida antarctica lipase B (CALB) has been compared in a continuous solid-gas reactor and in an organic liquid medium. The enthalpic and entropic contributions of water to the Gibbs free energy of activation in the gas phase were different from the ones in the organic phase, the inverse trends being observed for the variation of both DeltaH* and DeltaS* with water activity.Different phenomena were identified for their influence on the thermodynamic parameters. When increasing a(w), the enhanced flexibility of the enzyme was predominant in the gas phase whereas substrate-solvent interactions due to an increased polarity of the solvent affected mainly the thermodynamic parameters in the organic phase. The observed variations of DeltaG* with water activity were in accordance with kinetics results previously obtained in both reaction media.     

A pseudo steady‐state model for galacto‐oligosaccharides synthesis with β‐galactosidase from Aspergillus oryzae

A pseudo steady‐state model for the kinetically controlled synthesis of galacto‐oligosaccharides (GOS) with Aspergillus oryzae β‐galactosidase is presented. The model accounts for the dynamics of lactose consumption and production of galactose, glucose, di, tri, tetra, and penta‐oligosaccharides during the synthesis, being able to describe the total GOS content in the reaction medium at the experimental conditions evaluated. Experimental results show that the formation of GOS containing only galactose residues is significant at high conversions of substrate, which was taken into account in the model. The formation of enzyme transition complexes was considered and reasonable assumptions were made to reduce the number of parameters to be determined. The model developed has 8 parameters; 2 of them were experimentally determined and the other 6 were estimated by fitting to the experimental data using multiresponse regression. Temperature effect on kinetic and affinity constants was determined in the range from 40 to 55°C, and the data were fitted to Arrhenius type equation. Parameters of the proposed model are independent from the enzyme load in the reaction medium and, differently from previously reported models, they have a clear biochemical meaning. The magnitude of the kinetic and affinity constants of the enzyme suggests that the liberation of galactose from the galactosyl–enzyme complex is a very slow reaction and such complex is driven into GOS formation. It also suggests that the affinity for sugars of the galactosyl–enzyme complex is higher than that of the free enzyme. Biotechnol. Bioeng. 2011;108: 2270–2279. © 2011 Wiley Periodicals, Inc.     

A new mechanism and kinetic model for the enzymatic synthesis of short-chain fructooligosaccharides from sucrose

A kinetic model based on a ping-pong mechanism was developed under the steady-state hypothesis to account for the short-chain fructooligosaccharides (sc-FOS) synthesis using the commercial cellulolytic enzyme preparation, Rohapect CM. This new mechanism takes into account the interactions between the enzyme species and potential substrates (sucrose and sc-FOS) as a single complex reaction, allowing a better understanding of the reaction kinetics.The initial reaction rate laws appropriately describe the kinetic profiles of the examined substrates. Whereas sucrose exhibited Michaelis–Menten behavior with substrate inhibition, 1-kestose and nystose followed Michaelis–Menten and sigmoid enzyme kinetics. In addition, the enzyme was competitively inhibited by glucose and exhibited significant hydrolytic activity in the presence of nystose.The overall model was simultaneously fitted to experimental data from three initial sucrose concentrations (0.5, 1.5 and 2.1 M) using a multi-response regression with kinetic parameters that have biochemical relevance and are independent of the enzyme concentration. According to the model, sucrose acts almost exclusively as a fructosyl donor substrate. The mathematical development described herein is expected to be suitable for modeling similar enzymatic reaction systems.     

Rational polynomial equation as an unbiased approach for the kinetic studies of Drosophila melanogaster acetylcholinesterase reaction mechanism

The hydrolysis of substrates by cholinesterases does not follow the Michaelis–Menten reaction mechanism. The well-known inhibition by excess substrate is often accompanied by an unexpectedly high activity at low substrate concentrations. It appears that these peculiarities are the consequence of an unusual architecture of the active site, which conducts the substrate molecule over many stages before it is cleaved and released. Structural and kinetic data also suggest that two substrate molecules can attach at the same time to the free, as well as to the acetylated, enzyme. We present a procedure which provides an unbiased framework for mathematical modelling of such complex reaction mechanisms. It is based on regression analysis of a rational polynomial using classical initial rate data. The determination of polynomial degree reveals the number of independent parameters that can be evaluated from the available information. Once determined, these parameters can substantially facilitate the construction and evaluation of a kinetic model reflecting the expected molecular events in an enzymic reaction. We also present practical suggestions for testing the postulated kinetic model, using an original thermodynamic approach and an isolated effect in a specifically mutated enzyme.     

Highly efficient enzymatic synthesis of an ascorbyl unstaturated fatty acid ester with ecofriendly biomass‐derived 2‐methyltetrahydrofuran as cosolvent

Enzymatic synthesis of ascorbyl undecylenate, an unsaturated fatty acid ester of ascorbic acid, was reported with biomass‐derived 2‐methyltetrahydrofuran (MeTHF) as the cosolvent. Of the immobilized lipases tested, Candida antarctica lipase B (CAL‐B) showed the highest activity for enzymatic synthesis of ascorbyl undecylenate. Effect of reaction media on the enzymatic reaction was studied. The cosolvent mixture, t‐butanol‐MeTHF (1:4, v/v) proved to be the optimal medium, in which not only ascorbic acid had moderate solubility, but also CAL‐B showed a high activity, thus addressing the major problem of the solvent conflict for dissolving substrate and keeping satisfactory enzyme activity. In addition, the enzyme was much more stable in MeTHF and t‐butanol‐MeTHF (1:4) than in previously widely used organic solvents, t‐butanol, 2‐methyl‐2‐butanol, and acetone. The much higher initial reaction rate in this cosolvent mixture may be rationalized by the much lower apparent activation energy of this enzymatic reaction (26.6 vs. 38.1–39.1 kJ/mol) and higher enzyme catalytic efficiency (V_max/K_m, 8.4 vs. 1.3–1.4 h^?1). Ascorbyl undecylenate was obtained with the yields of 84–89% and 6‐regioselectivity of >99% in t‐butanol‐MeTHF (1:4) at supersaturated substrate concentrations (60 and 100 mM) after 5–8 h. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1005–1011, 2014     

Thermodynamically based solvent design for enzymatic saccharide acylation with hydroxycinnamic acids in non-conventional media

Enzyme-catalyzed synthesis has been widely studied with lipases (EC 3.1.1.3), but feruloyl esterases (FAEs; EC 3.1.1.73) may provide advantages such as higher substrate affinity and regioselectivity in the synthesis of hydroxycinnamate saccharide esters. These compounds are interesting because of their amphiphilicity and antioxidative potential. Synthetic reactions using mono- or disaccharides as one of the substrates may moreover direct new routes for biomass upgrading in the biorefinery. The paper reviews the available data for enzymatic hydroxycinnamate saccharide ester synthesis in organic solvent systems as well as other enzymatic hydroxycinnamate acylations in ionic liquid systems. The choice of solvent system is highly decisive for enzyme stability, selectivity, and reaction yields in these synthesis reactions. To increase the understanding of the reaction environment and to facilitate solvent screening as a crucial part of the reaction design, the review explores the use of activity coefficient models for describing these systems and - more importantly - the use of group contribution model UNIFAC and quantum chemistry based COSMO-RS for thermodynamic predictions and preliminary solvent screening. Surfactant-free microemulsions of a hydrocarbon, a polar alcohol, and water are interesting solvent systems because they accommodate different substrate and product solubilities and maintain enzyme stability. Ionic liquids may provide advantages as solvents in terms of increased substrate and product solubility, higher reactivity and selectivity, as well as tunable physicochemical properties, but their design should be carefully considered in relation to enzyme stability. The treatise shows that thermodynamic modeling tools for solvent design provide a new toolbox to design enzyme-catalyzed synthetic reactions from biomass sources.     

New insights into the catalytic mechanism of human glycine N‐acyltransferase

Even though the glycine conjugation pathway was one of the first metabolic pathways to be discovered, this pathway remains very poorly characterized. The bi‐substrate kinetic parameters of a recombinant human glycine N‐acyltransferase (GLYAT, E.C. 2.3.1.13) were determined using the traditional colorimetric method and a newly developed HPLC–ESI‐MS/MS method. Previous studies analyzing the kinetic parameters of GLYAT, indicated a random Bi–Bi and/or ping‐pong mechanism. In this study, the hippuric acid concentrations produced by the GLYAT enzyme reaction were analyzed using the allosteric sigmoidal enzyme kinetic module. Analyses of the initial rate (v) against substrate concentration plots, produced a sigmoidal curve (substrate activation) when the benzoyl‐CoA concentrations was kept constant, whereas the plot with glycine concentrations kept constant, passed through a maximum (substrate inhibition). Thus, human GLYAT exhibits mechanistic kinetic cooperativity as described by the Ferdinand enzyme mechanism rather than the previously assumed Michaelis–Menten reaction mechanism.     

Adsorptive control of water in esterification with immobilized enzymes: II. fixed-bed reactor behavior

Experimental and theoretical studies are conducted to understand the dynamic behavior of a continuous-flow fixed-bed reactor in which an esterification is catalyzed by an immobilized enzyme in an organic solvent medium. The experimental system consists of a commercial immobilized lipase preparation known as Lipozyme as the biocatalyst, with propionic acid and isoamyl alcohol (dissolved in hexane) as the reaction substrates. A complex dynamic behavior is observed experimentally as a result of the simultaneous occurrence of reaction and adsorption phenomena. Both propionic acid and water are adsorbed by the biocatalyst resulting in lower reaction rates. In addition, an excessive accumulation of water in the reactor leads to a rapid irreversible inactivation of the enzyme. A model based on previously-obtained adsorption isotherms and kinetic expressions, as well as on adsorption rate measurements obtained in this work, is used to predict the concentration and thermodynamic activity of water along the reactor length. The model successfully predicts the dynamic behavior of the reactor and shows that a maximum thermodynamic activity of water occurs at a point at some distance from the reactor entrance. A cation exchange resin in sodium form, packed in the reactor as a selective water adsorbent together with the catalyst particles, is shown to be an effective means for preventing an excessive accumulation of water formed in the reaction. Its use results in longer cycle times and greater productivity. As predicted by the model, the experimental results show that the water adsorbed on the catalyst and on the ion exchange resin can be removed with isoamyl alcohol with no apparent loss in enzyme activity.     

Enzyme reaction kinetics in organic solvents: A theoretical kinetic model and comparison with experimental observations

A theoretical kinetic model has been developed in order to describe the enzyme reaction in organic solvents. In this model the hydration of the enzyme molecule was examined and the equilibrium kinetic constants expressed in terms of thermodynamic activity. Analysis of a proposed kinetic model shows that the enzyme reaction rate in organic solvents is determined by two factors: substrate solvation and enzyme hydration, which are determined by the activity coefficient of the substrate and the water activity of the reaction media, respectively. The activity coefficient of the substrate and the water activity have been calculated using the UNIFAC equation to analyze the effects of organic solvents on the rate of enzyme reaction, and the results were compared with experimental data. Predictions of the proposed model were found to be in good agreement with previous experimental observations.     

Direct enantioselective HPLC monitoring of lipase‐catalyzed kinetic resolution of flurbiprofen

The solvent versatility of Chiralpak IB, a 3,5‐dimethylphenylcarbamate derivative of cellulose‐based chiral stationary phase, is demonstrated in the direct enantioselective HPLC monitoring of lipase‐catalyzed kinetic resolution of flurbiprofen in nonstandard HPLC organic solvents. Nonstandard HPLC organic solvents were used as the reaction media for the lipase‐catalysis and in mean time as diluent to dissolve the “difficult to dissolve” enzyme substrate (the acid) and as eluent for the simultaneous enantioselective HPLC baseline separation of both substrate and product in one run without any further derivatization. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Modeling of uncertainties in biochemical reactions

Mathematical modeling is an indispensable tool for research and development in biotechnology and bioengineering. The formulation of kinetic models of biochemical networks depends on knowledge of the kinetic properties of the enzymes of the individual reactions. However, kinetic data acquired from experimental observations bring along uncertainties due to various experimental conditions and measurement methods. In this contribution, we propose a novel way to model the uncertainty in the enzyme kinetics and to predict quantitatively the responses of metabolic reactions to the changes in enzyme activities under uncertainty. The proposed methodology accounts explicitly for mechanistic properties of enzymes and physico‐chemical and thermodynamic constraints, and is based on formalism from systems theory and metabolic control analysis. We achieve this by observing that kinetic responses of metabolic reactions depend: (i) on the distribution of the enzymes among their free form and all reactive states; (ii) on the equilibrium displacements of the overall reaction and that of the individual enzymatic steps; and (iii) on the net fluxes through the enzyme. Relying on this observation, we develop a novel, efficient Monte Carlo sampling procedure to generate all states within a metabolic reaction that satisfy imposed constrains. Thus, we derive the statistics of the expected responses of the metabolic reactions to changes in enzyme levels and activities, in the levels of metabolites, and in the values of the kinetic parameters. We present aspects of the proposed framework through an example of the fundamental three‐step reversible enzymatic reaction mechanism. We demonstrate that the equilibrium displacements of the individual enzymatic steps have an important influence on kinetic responses of the enzyme. Furthermore, we derive the conditions that must be satisfied by a reversible three‐step enzymatic reaction operating far away from the equilibrium in order to respond to changes in metabolite levels according to the irreversible Michelis–Menten kinetics. The efficient sampling procedure allows easy, scalable, implementation of this methodology to modeling of large‐scale biochemical networks. Biotechnol. Bioeng. 2011;108: 413–423. © 2010 Wiley Periodicals, Inc.     

Esterification by immobilized lipase in solvent-free media: kinetic and thermodynamic arguments

The aim of this study is to characterize, in solvent-free systems (SFS), the kinetic and thermodynamic performance of batch lipase-catalyzed esterification. SFS are compared to a conventional organic solvent, n-hexane. The esterification of oleic acid with ethanol was chosen as a model reaction. The TABEK (thermodynamic activity-based enzyme kinetics) approach was used to rationally analyze kinetics. Influence of the reaction medium on final conversions was also studied. Several factors, such as initial molar ratio of substrates, reactant availability, initial water content, and quantity of immobilized enzyme, were examined. Special attention was also turned to enzyme stability and reuse after reaction, this last item being a prerequisite in the development of industrial processes. SFS proved to be almost as efficient as n-hexane from a kinetic and thermodynamic point of view and offered a better volumetric production.     

Thermodynamic Constraints in Kinetic Modeling: Thermodynamic‐Kinetic Modeling in Comparison to Other Approaches

Kinetic models of reaction networks may easily violate the laws of thermodynamics and the principle of detailed balance. In large network models, the constraints that are imposed by these laws are particularly difficult to address. This hinders modeling of biochemical reaction networks. Thermodynamic‐kinetic modeling is a method that provides a thermodynamically sound and formally appealing way for deriving dynamic model equations of reaction systems. State variables of this approach are thermokinetic potentials that describe the ability of compounds to drive a reaction. A compound has a parameter called capacity, which is the ratio of its concentration and thermokinetic potential. A reaction is described by its resistance which is the ratio of the thermokinetic driving force and flux. In these aspects, the formalism is similar to the modeling formalism for electrical networks and an analogous graphical representation is possible. The thermodynamic‐kinetic modeling formalism is equivalent to the traditional kinetic modeling formalism with the exception that it is not possible to build thermodynamically infeasible models. Here, the thermodynamic‐kinetic modeling formalism is reviewed, compared to other approaches, and some of its advantages are worked out. In contrast to other approaches, thermodynamic‐kinetic modeling does not rely on an explicit enumeration of stoichiometric cycles. It is capable of describing rate laws far from equilibrium. Further, the parameterization by capacities and resistances is particularly intuitive and powerful.     

Expression and characterization of Pichia etchellsii β-glucosidase in Escherichia coli

The β-glucosidase enzyme is important as the terminal enzyme involved in hydrolysis of cellobiose and short-chain cellodextrins generated during enzymatic cellulose degradation. Under controlled reaction conditions the enzyme also displays cello-oligosaccharide synthesizing ability (based on either the thermodynamic or kinetic approach). We present here the purification of the enzyme β-glucosidase (BGL) of Pichia etchellsii from recombinant pBG55 Escherichia coli clone. The kinetic parameters, substrate specificity and oligosaccharide synthesizing ability of the purified enzyme are also reported. The purified 200-kDa protein (tetramer of 50 kDa) was identified as a broad-substrate-specificity enzyme exhibiting increased temperature and glucose tolerance compared to the native yeast enzyme. Temperature directed substrate specificity for aryl β,1–4 linkage, and β(1–2), β(1–4), β(1–6) and β(2-1) linkages in various natural disaccharides was observed. Glycosylation of the enzyme was found to be unimportant for enzyme activity. With both cellobiose and glucose, oligosaccharide synthesis was detected. The implications of this information with regard to cellulose hydrolysis and oligosaccharide synthesis are discussed.     

Kinetic modeling of lipase‐catalyzed esterification reaction between oleic acid and trimethylolpropane: A simplified model for multi‐substrate multi‐product ping–pong mechanisms

Kinetic models are among the tools that can be used for optimization of biocatalytic reactions as well as for facilitating process design and upscaling in order to improve productivity and economy of these processes. Mechanism pathways for multi‐substrate multi‐product enzyme‐catalyzed reactions can become very complex and lead to kinetic models comprising several tens of terms. Hence the models comprise too many parameters, which are in general highly correlated and their estimations are often prone to huge errors. In this study, Novozym^®435 catalyzed esterification reaction between oleic acid (OA) and trimethylolpropane (TMP) with continuous removal of side‐product (water) was carried out as an example for reactions that follow multi‐substrate multi‐product ping‐pong mechanisms. A kinetic model was developed based on a simplified ping‐pong mechanism proposed for the reaction. The model considered both enzymatic and spontaneous reactions involved and also the effect of product removal during the reaction. The kinetic model parameters were estimated using nonlinear curve fitting through unconstrained optimization methodology and the model was verified by using empirical data from different experiments and showed good predictability of the reaction under different conditions. This approach can be applied to similar biocatalytic processes to facilitate their optimization and design. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1422–1429, 2013     

Why do crown ethers activate enzymes in organic solvents?

One of the major drawbacks of enzymes in nonaqueous solvents is that their activity is often dramatically low compared to that in water. This limitation can be largely overcome by crown ether treatment of enzymes. In this paper, we describe a number of carefully designed new experiments that have improved the insights into the mechanisms that are operative in the crown ether activation of enzymes in organic solvents. The enhancement of enzyme activity upon addition of 18-crown-6 to the organic solvent can be reconciled with a mechanism in which macrocyclic interactions of 18-crown-6 with the enzyme play an important role. Macrocyclic interactions (e.g., complexation with lysine ammonium groups of the enzyme) can lead to a reduced formation of inter- and intramolecular salt bridges and, consequently, to lowering of the kinetic conformational barriers, enabling the enzyme to refold into thermodynamically stable, catalytically (more) active conformations. This assumption is supported by the observation that the crown-ether-enhanced enzyme activity is retained after removal of the crown by washing with a dry organic solvent. A much stronger crown ether activation is observed when 18-crown-6 is added prior to lyophilization, and this can be explained by a combination of two effects: the before-mentioned macrocyclic complexation effect, and a less specific, nonmacrocyclic, lyoprotecting effect. The magnitude of the total crown ether effect depends on the polarity and thermodynamic water activity of the solvent, the activation being highest in dry and apolar media, where kinetic conformational barriers are highest. By determination of the specific activity of crown-ether-lyophilized enzyme as a function of the enzyme concentration, the macrocyclic crown ether (linearly dependent on the enzyme concentration) and the nonmacrocyclic lyoprotection effect (not dependent on the enzyme concentration) could be separated. These measurements reveal that the contribution of the nonmacrocyclic effect is significantly larger than the macrocyclic refolding effect.     

Solvent selection and optimization of α‐chymotrypsin‐catalyzed synthesis of N‐Ac‐Phe‐Tyr‐NH2 using mixture design and response surface methodology

A peptide, N‐Ac‐Phe‐Tyr‐NH₂, with angiotensin I‐converting enzyme (ACE) inhibitor activity was synthesized by an α‐chymotrypsin‐catalyzed condensation reaction of N‐acetyl phenylalanine ethyl ester (N‐Ac‐Phe‐OEt) and tyrosinamide (Tyr‐NH₂). Three kinds of solvents: a Tris–HCl buffer (80 mM, pH 9.0), dimethylsulfoxide (DMSO), and acetonitrile were employed in this study. The optimum reaction solvent component was determined by simplex centroid mixture design. The synthesis efficiency was enhanced in an organic‐aqueous solvent (Tris‐HCl buffer: DMSO: acetonitrile = 2:1:1) in which 73.55% of the yield of N‐Ac‐Phe‐Tyr‐NH₂ could be achieved. Furthermore, the effect of reaction parameters on the yield was evaluated by response surface methodology (RSM) using a central composite rotatable design (CCRD). Based on a ridge max analysis, the optimum condition for this peptide synthesis included a reaction time of 7.4 min, a reaction temperature of 28.1°C, an enzyme activity of 98.9 U, and a substrate molar ratio (Phe:Tyr) of 1:2.8. The predicted and the actual (experimental) yields were 87.6 and 85.5%, respectively. The experimental design and RSM performed well in the optimization of synthesis of N‐Ac‐Phe‐Tyr‐NH₂, so it is expected to be an effective method for obtaining a good yield of enzymatic peptide. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Chemically reprogramming the phospho‐transfer reaction to crosslink protein kinases to their substrates

The proteomic mapping of enzyme–substrate interactions is challenged by their transient nature. A method to capture interacting protein kinases in complexes with a single substrate of interest would provide a new tool for mapping kinase signaling networks. Here, we describe a nucleotide‐based substrate analog capable of reprogramming the wild‐type phosphoryl‐transfer reaction to produce a kinase‐acrylamide‐based thioether crosslink to mutant substrates with a cysteine nucleophile substituted at the native phosphorylation site. A previously reported ATP‐based methacrylate crosslinker (ATP‐MA) was capable of mediating kinase crosslinking to short peptides but not protein substrates. Exploration of structural variants of ATP‐MA to enable crosslinking of protein substrates to kinases led to the discovery that an ADP‐based methacrylate (ADP‐MA) crosslinker was superior to the ATP scaffold at crosslinking in vitro. The improved efficiency of ADP‐MA over ATP‐MA is due to reduced inhibition of the second step of the kinase–substrate crosslinking reaction by the product of the first step of the reaction. The new probe, ADP‐MA, demonstrated enhanced in vitro crosslinking between the Src tyrosine kinase and its substrate Cortactin in a phosphorylation site‐specific manner. The kinase–substrate crosslinking reaction can be carried out in a complex mammalian cell lysate setting, although the low abundance of endogenous kinases remains a significant challenge for efficient capture.     

Enzyme activity evaluation of organic solvent-treated phenylalanine ammonia lyase

The direct one-step synthesis of L-phenylalanine methyl ester in an organic-aqueous biphasic system using phenylalanine ammonia lyase (E.C.4.3.1.5, PAL) containing Rhodotorula glutinis yeast whole cells was reported earlier. We report here further optimization of this biotransformation using isolated PAL, when the lyophilized enzyme is treated with different water miscible and water immiscible organic solvents. Use of isolated PAL enzyme is advantageous in overcoming diffusion barriers encountered when using PAL containing R.glutinis whole cells, and resulted in increased product yield due to better interaction of enzyme with the substrate. Among the water miscible solvents, ethanol treated and methanol-treated enzymes supported maximum PAL forward and reverse activities; respectively. In the water immiscible solvents category, heptane-treated enzyme exhibited maximal activity for both PAL forward and reverse reactions. PAL activity obtained with enzyme specimens treated with methanol, ethanol, and heptane varied in the range of 91–99% of that observed in aqueous buffer medium for the forward reaction; and 89–95% for the reverse reaction. n-butanol,acetone, and benzene were found to have a inhibitory effect on PAL enzyme, in that, it resulted in only 31–33% activity of that obtained with aqueous solution. Raman spectroscopy was used to monitor amide I and II bands which are sensitive to changes in the secondary structure of proteins. No changes in structure could be detected from the analyses of AI and AII bands of PAL spectra. This data obtained for PAL, a tetramer, could be significant in predicting how solvent interactions affect the structure and function of multimeric proteins and enzymes in nonaqueous media.