On the importance of the support material for bioorganic synthesis. Influence of water partition between solvent, enzyme and solid support in water-poor reaction media

alpha-Chymotrypsin was adsorbed on solid support materials and the catalytic activity of the preparations in organic solvents was studied. The activity was highly dependent on the nature of the support material and on the amount of water present in the reaction mixture. There appears to be competition for the water in the system between the enzyme, the support material and the solvent. The support materials were characterized by measuring their ability to absorb water from water-saturated diisopropyl ether. For the quotient: (amount of water on the support)/(amount of water in the solvent) in the model system the term aquaphilicity was proposed. The activity of adsorbed chymotrypsin in diisopropyl ether decreased with increasing aquaphilicity of the support material. The same trend was observed when the activity of horse liver alcohol dehydrogenase adsorbed on different supports was measured in diisopropyl ether.     

Designing enzymatic kyotorphin synthesis in organic media with low water content

Design of enzymatic kyotorphin synthesis in low water media has been carried out as a function of enzyme nature, the immobilization support material and the reaction medium, by using N-benzoyl-L-tyrosine ethyl ester and L-argininamide as substrates. Native and chemically-glycated alpha-chymotrypsin deposited on supports with different degrees of aquaphilicity (celite, polypropylene PP, and polyamide PA6) were used as catalysts. Binary organic solvent systems of ethanol and different water-immiscible organic cosolvents (ethylacetate, tert-butanol, chloroform, toluene, n-hexane, and n-octane) were studied as reaction media at constant water content (3% v/v). The greater the water binding affinity of the support the lower the synthetic activity of deposited enzymes: the activity of the celite derivative was 4x greater than the polyamide derivative. The enzyme glycation process hardly modified the catalytic ability of the celite derivative, but resulted in a moderate increase in operational stability. The presence of hydrophobic organic cosolvents in the water/ethanol reaction medium significantly increased enzyme activity, whereas the selectivity of the reaction remained high. Hexane was shown to be the best cosolvent, the synthetic activity of the celite derivative in hexane-ethanol (77 : 20%, v/v) being 130x greater than that in 97% (v/v) ethanol.     

Effect of support material and enzyme pretreatment on enantioselectivity of immobilized subtilisin in organic solvents

Subtilisin Carlsberg was covalently attached to five macroporous acrylic supports of varying aquaphilicity (a measure of hydrophilicity). Kinetic parameters of the transesterification of S and R enantiomers of secphenethyl alcohol with vinyl butyrate, catalyzed by various immobilized subtilisins, were determined in anhydrous dioxane and acetonitrile. Enzyme enantioselectivity in acetonitrile, but not in dioxane, correlated with the aquaphilicity of the support; a mechanistic rationale for this phenomenon was proposed. Although the catalytic activity of immobilized subtilisin in anhydrous solvents strongly depended on enzyme pretreatment, the enantioselectivity was essential conserved. (c) 1994 John Wiley & Sons, Inc.     

The effects of reformulating buffer species and their concentrations on subtilisin-catalyzed optical resolution of racemic 1-phenylethylamine in 3-methyl-3-pentanol

For the optical resolution of racemic 1-phenylethylamine in 3-methyl-3-pentanol, subtilisin was reformulated by lyophilization with buffer salts. The amide synthesis activity of subtilisin in organic solvent was compared with the hydrolysis activity in aqueous buffer when different buffer species and their concentrations were used in lyophilization. The enzyme activity in organic solvent showed a different pattern from that of the hydrolysis depending upon the species and the concentrations of buffers. Morphology of the reformulated subtilisin was examined by scanning electron microscopy (SEM). The porosity of reformulated subtilisin particles increased up to the optimal buffer concentrations for the amide synthesis in organic solvent. Glassy looks and decrease in porosity developed at high (i.e. above the optimal) buffer concentrations appear to affect the decrease in the synthetic activity in organic media.     

On the importance of the support material for enzymatic synthesis in organic media. Support effects at controlled water activity

Enzymes were deposited on different porous support materials and these preparations were used to catalyze reactions in organic media. Reactions were carried out at specific water activities, achieved by equilibrating both the enzyme preparation and the substrate solution at the desired water activity before mixing them and thereby starting the reactions. The reaction rates obtained at the same water activity with different supports differed greatly, indicating a direct influence of the support on the enzyme. For horse liver alcohol dehydrogenase, Celite was the best support, and the reaction rate increased with increasing water activity. In the alpha-chymotrypsin-catalyzed alcoholysis of N-acetyl-L-phenylalanine ethyl ester with 1-butanol, high rates were again obtained with Celite, but with this support only about one third of the ethyl ester was converted to butyl ester, the rest was hydrolyzed. With the polyamide support, Accurel PA6, alcoholysis was the dominating reaction, and by using a low water activity (0.33), hydrolysis was completely suppressed while still maintaining a high alcoholysis activity. Controlled pore glass (CPG), derivatized with either hexyl or glucosyl groups, had quite different properties as enzyme supports. For horse liver alcohol dehydrogenase, glucose-CPG was a much better support than hexyl-CPG, and in the alpha-chymotrypsin-catalyzed reactions, glucose-CPG favored hydrolysis, and hexyl-CPG alcoholysis, at water activities exceeding 0.8. The results are discussed considering the absorption of water on the enzymes, on the supports and the solubility of water in the reaction media; all these parameters were measured separately.     

Optimization of methyl propionate production catalysed by Mucor miehei lipase

Lipase from Mucor miehei was used to catalyse the esterification reaction between propionic acid and methyl alcohol in modified organic media. Small-scale model studies were performed in order to define the optimal conditions. The specific activity of immobilized lipase, adsorbed onto hydrophilic supports, compared to free lipase, showed that enzyme activity was altered by immobilisation. Non-polar solvents were shown to be less harmful for the biocatalyst than solvents with higher polarity. Diethyl ether was used as the cosolvent of hexane to improve the solubility of substrates in the organic phase thus increasing contact with enzyme. An optimal ratio of 90/10 (v/v) was determined for a hexane/diethyl ether mixture. The mass of enzyme preparation must be high enough to display optimal diffusion of the reagents and hydration of the catalytic sites. Increased substrate concentrations were stimulatory up to a point after which inhibition and enzyme destabilisation, in repeated runs, occurred. Water saturation of the organic medium greatly lowered the biosynthetic activity of the enzyme. It was possible to reach a 96% methyl propionate biosynthesis yield after 2.30 h reaction, underlining the free-enzyme operational capacity in a quasi-anhydrous modified organic medium.     

Influence of Water Activity and Support Material on the Enzymatic Synthesis of a Cck-8 Tripeptide Fragment

The kinetically controlled condensation of Z-Gly-Trp-OMe and H-Met-OEt catalyzed by α-chymo-trypsin in organic media is reported. The influence of thermodynamic water activity and the support material used to adsorb α-chymotrypsin, on both the product yield and enzymatic activity was investigated. Polyamide based materials were the best support at low water activity rendering the highest reaction rates and yields. The activity of the adsorbed enzyme at low water activities depends on both the accessible surface area and the hydrophobicity of the support. Polyamide had both adequate hydrophilicity and high surface area yielding the best results. Polypropylene based supports were strongly hydrophobic and, although they presented a high surface area, the enzymatic activity was much lower. The solvents used to carry out the synthesis were acetonitrile and ethyl acetate. No significant differences were observed on the performance of the reaction in either solvent. The tripeptide selected is a fragment of the cholecystokinin C-terminal octapeptide (CCK-8), a biological active peptide involved in the control of gastrointestinal function.     

Optimizing the salt-induced activation of enzymes in organic solvents: effects of lyophilization time and water content

The addition of simple inorganic salts to aqueous enzyme solutions prior to lyophilization results in a dramatic activation of the dried powder in organic media relative to enzyme with no added salt. Activation of both the serine protease subtilisin Carlsberg and lipase from Mucor javanicus resulting from lyophilization in the presence of KCl was highly sensitive to the lyophilization time and water content of the sample. Specifically, for a preparation containing 98% (w/w) KCl, 1% (w/w) phosphate buffer, and 1% (w/w) enzyme, varying the lyophilization time showed a direct correlation between water content and activity up to an optimum, beyond which the activity decreased with increasing lyophilization time. The catalytic efficiency in hexane varied as much as 13-fold for subtilisin Carlsberg and 11-fold for lipase depending on the lyophilization time. This dependence was apparently a consequence of including the salt, as a similar result was not observed for the enzyme freeze-dried without KCl. In the case of subtilisin Carlsberg, the salt-induced optimum value of kcat/Km for transesterification in hexane was over 20,000-fold higher than that for salt-free enzyme, a substantial improvement over the previously reported enhancement of 3750-fold (Khmelnitsky, 1994). As was found previously for pure enzyme, the salt-activated enzyme exhibited greatest activity when lyophilized from a solution of pH equal to the pH for optimal activity in water. The active-site content of the lyophilized enzyme samples also depended upon lyophilization time and inclusion of salt, with opposite trends in this dependence observed for the solvents hexane and tetrahydrofuran. Finally, substrate selectivity experiments suggested that mechanism(s) other than selective partitioning of substrate into the enzyme-salt matrix are responsible for salt-induced activation of enzymes in organic solvents.     

Efficient production of diltiazem chiral intermediate using immobilized lipase from <Emphasis Type="Italic">Serratia marcescens</Emphasis>

The lipase from Serratia marcescence ECU1010 (Sml) was capable of enantioselectively catalyzing the synthesis of many chiral drug precursors. This paper investigated the immobilization of Sml on appropriate supporting materials and its performance in bioreactor. Chitosan, Celite 545, and DEAE-cellulose were found to be the ideal supports among 8 carriers tested with respect to enzyme load and activity recovery of lipase. When Sml was immobilized, significant improvements of stability against pH, thermal, and operational deactivation were observed with all the 3 better supports, and the best stability was observed when the lipase was immobilized on glutaraldehyde activated chitosan. As for the effect of organic solvent in the biphasic reaction system, the hydrolytic activity of the immobilized lipase on trans-3-(4′-methoxyphenyl)glycidic acid methyl ester ((±)-MPGM) observed in isopropyl ether was 6 and 3 times higher than those in toluene and methyl tert-butyl ether. The lipasecatalyzed production of (−)-MPGM by enzymatic resolution of (±)-MPGM with chitosan-Sml in isopropyl etherwater biphasic system was carried out in a 2 L stirred-tank reactor. The batch operation was more efficient operation mode for the enantioselective hydrolysis of (±)-MPGM, affording enantiopure (−)-MPGM in 44.3% overall yield, in contrast to 29.3% in a continuous reactor.     

Hydrogen/deuterium exchange study of subtilisin Carlsberg during prolonged exposure to organic solvents

It has been previously reported that prolonged exposure of an enzyme to organic solvents leads to substantial decrease of activity. This effect was found to be unrelated to the catalysts' structure or their possible aggregation in organic solvents, and up to the present day the cause for activity loss remains unclear. In the present work, the structural dynamics of the serine protease subtilisin Carlsberg (SC) have been investigated during prolonged exposure to two organic solvents by following hydrogen/deuterium (H/D) exchange of mobile protons. The enzyme, after lyophilization, was incubated in organic solvents at controlled deuteriated water activity for different times and the H/D exchange was allowed to take place. The amount of deuterium exchanged was evaluated by (2)H NMR, which in turn gave us a picture of the changing dynamics of our model enzyme during incubation and under different experimental conditions. Our results show that the flexibility of SC decreases during prolonged storage in 1,4-dioxane (Diox) and acetonitrile (ACN) as indicated by the observed 3- to 10-fold decrease in the apparent rate constants of exchange (k) of fast exchangeable protons (FEP) and slow exchangeable protons (SEP) in the protein. Our study also shows that SC is more flexible in ACN than in Diox (k 3-20 times higher in ACN for the FEP and SEP), suggesting that enzyme dynamics are affected by solvent physicochemical properties. Additionally, the enzyme dynamics are also affected by the method of preparation: decreased flexibility (k decreases 3- to 10-fold for FEP and SEP) is observed when the enzyme is chemically modified with poly ethylene glycol (PEGylated) or colyophilized with crown ethers. A possible relationship between activity, enantioselectivity (E), and structural dynamics is discussed, demonstrating that direct correlations, as have been attempted in the past, are hampered by the multi-variable nature and complexity of the system.     

Variations in the enantioselectivity of salt-activated subtilisin induced by lyophilization

Including excess salt during lyophilization has been shown to increase the activity of freeze-dried subtilisin Carlsberg (SC) in anhydrous media by over 20,000-fold [Ru et al. (1999) Biotechnol Bioeng 63:233-241]. In the present study, salt-activated SC (KCl-SC) showed a 30% enhancement in enantioselectivity compared to the salt-free enzyme in a variety of organic solvents. Activity toward both enantiomers of N-acetyl-phenylalanine methyl ester (APME) increased in tandem by 2-3 orders of magnitude in all solvents, indicating that the mechanism of salt activation is inherent to the enzyme and does not strongly favor one enantiomer over the other. However, activity and enantioselectivity of salt-activated SC could be manipulated through changes in the lyophilization conditions. Variations in lyophilization time, initial KCl concentration, and initial lyophilization volume altered enantioselectivity over 2-fold. The changes in enantioselectivity reflected the activity for the L enantiomer, while the activity toward the D enantiomer was mostly unaffected. The results indicate that the lyophilization time and final water content of the KCl-SC are important determinants of enzyme activity for the L enantiomer, suggesting that the favored reaction is more sensitive to the structural integrity of the salt-activated enzyme.     

FTIR-ATR characterization of free Rhizomucor meihei lipase (RML), Lipozyme RM IM and chitosan-immobilized RML

The synthesis and characterization of biocatalysts based on lipase from Rhizomucor miehei (RML) immobilized on chitosan-based supports were investigated. The enzyme was immobilized on chitosan following two strategies: (i) physical adsorption; and (ii) covalent bonding using glutaraldehyde. The content of enzyme bound in the supports, as precipitable protein, was analyzed using UV/visible methods. FTIR-ATR spectroscopy was employed to characterize the prepared biocatalysts, as well the native enzyme and a commercial biocatalyst Lipozyme RM IM, used as reference materials. Analysis of the amide I′ signal allowed to follow the changes in the secondary structure of the enzyme after binding to the support and its thermal stability. The hydrolysis of ethyl stearate monitored in situ by FTIR-ATR was used as a test reaction. Results showed that RML was bound to Chit and Glut–Chit with minor changes in its secondary structure, thermal stability and enzymatic activity in a selected reaction test.     

Activity and stability of cross-linked tyrosinase aggregates in aqueous and nonaqueous media

Cross-linked tyrosinase aggregates were prepared by precipitating the enzyme with ammonium sulfate and subsequent cross-linking with glutaraldehyde. Both activity and stability of these cross-linked enzyme aggregates (CLEAs) in aqueous solution, organic solvents, and ionic liquids have been investigated. Immobilization effectively improved the stability of the enzyme in aqueous solution against various deactivating conditions such as pH, temperature, denaturants, inhibitors, and organic solvents. The stability of the CLEAs in various organic solvents such as tert-butanol (t(1/2)=326.7h at 40°C) was significantly enhanced relative to that in aqueous solution (t(1/2)=5.5h). The effect of thermodynamic water activity (a(w)) on the CLEA activity in organic media was examined, demonstrating that the enzyme incorporated into CLEAs required an extensive hydration (with an a(w) approaching 1.0) for optimizing its activity. The impact of ionic liquids on the CLEA activity in aqueous solution was also assessed.     

Catalytic and structural properties of surfactant-horseradish peroxidase complex in organic media

A surfactant-horseradish peroxidase (HRP) complex that is catalytically active in organic media has been successfully prepared by a method utilizing water-in-oil (W/O) emulsions. To optimize conditions for preparation of the HRP complex, the effects of some key parameters in the aqueous phase of W/O emulsions were investigated. The surfactant-HRP complex prepared with a nonionic surfactant exhibited a high catalytic activity compared to those with a cationic or anionic surfactant in anhydrous benzene. At the preparation step, the pH of the aqueous solution had a prominent effect on the enzymatic activity of the HRP complex in organic media. Several kinds of salts present in the HRP complex could be employed to enhance the catalytic performance in organic media. However, anionic ions present in the preparation process appeared to lower the catalytic activity owing to the complexation with heme iron. UV-visible absorption spectra of the HRP complex in benzene, which were prepared from a KCN solution (pH 7.0) or an alkaline solution (pH 12), were comparable with those of native HRP in aqueous solution under the same conditions. Resonance Raman spectroscopic studies also revealed that no significant change in the coordination state of the heme iron occurred even after coating the enzyme with surfactant molecules, lyophilization, and solubilization in nonaqueous media.     

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.     

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.     

Modeling hydration mechanisms of enzymes in nonpolar and polar organic solvents

A comprehensive study of the hydration mechanism of an enzyme in nonaqueous media was done using molecular dynamics simulations in five organic solvents with different polarities, namely, hexane, 3-pentanone, diisopropyl ether, ethanol, and acetonitrile. In these solvents, the serine protease cutinase from Fusarium solani pisi was increasingly hydrated with 12 different hydration levels ranging from 5% to 100% (w/w) (weight of water/weight of protein). The ability of organic solvents to 'strip off' water from the enzyme surface was clearly dependent on the nature of the organic solvent. The rmsd of the enzyme from the crystal structure was shown to be lower at specific hydration levels, depending on the organic solvent used. It was also shown that organic solvents determine the structure and dynamics of water at the enzyme surface. Nonpolar solvents enhance the formation of large clusters of water that are tightly bound to the enzyme, whereas water in polar organic solvents is fragmented in small clusters loosely bound to the enzyme surface. Ions seem to play an important role in the stabilization of exposed charged residues, mainly at low hydration levels. A common feature is found for the preferential localization of water molecules at particular regions of the enzyme surface in all organic solvents: water seems to be localized at equivalent regions of the enzyme surface independently of the organic solvent employed.     

Intramolecular esterification by lipase powder in microaqueous benzene: factors affecting activity of pure enzyme

Various factors affecting the catalytic activity of pure lipase of Pseudomonas fluorescens in microaqueous benzene were investigated with respect to lactonization of 15-hydroxypentadecanoic acid. Without deposition of the enzyme or of the enzyme plus activity enhancer (additive) on celite powder, the pure enzyme was very poorly dispersed in the microaqueous benzene, resulting in very low activity. The enzyme immobilized on celite powder exhibited the highest activity at a free water content of ca. 0.083%. When a sugar alcohol such as erythritol, arabitol, or sorbitol was added before lyophilization with approximate proportion of 3 g/g enzyme, marked increases in the enzyme activity were observed at a shifted optimal free water content, i.e., 0.04%. Inclusion of phosphotidylcholine resulted in a somewhat higher activity than in the system of enzyme plus celite only. Addition of lactose, bovine serum albumin, casein, dextran, polyvinyl alcohol, phosphate, or NaCl all caused a decrease in the enzyme activity. From the effects of the additives examined, it is deduced that the following three factors are required for a pure enzyme to exhibit its full activity in a water-immiscible organic solvent: (1) optimum moisture content, (2) disperser (support particles having enough surface area on which the enzyme is thinly deposited), and (3) activity enhancer (additive) at optimum concentration The importance of noting the purity of the enzyme preparation is emphasized when its catalysis in an organic solvent is investigated.     

The activity of human CYP2D6 in low water organic solvents

P450 enzymes are of high interest for synthetic applications due to their ability to catalyze hydroxylation reactions at inactivated C-H bonds. The low solubility of many substrates in buffer, however, is limiting the applications of P450s. Our recent demonstration that the P450 enzymes CYP2D6 and CYP3A4 can function very well in biphasic solvent systems is one step towards overcoming this drawback, but is not practical when substrates or products are unstable in water, or with water-soluble products. An alternative strategy, which also facilitates enzyme recycling, is to directly resuspend lyophilized enzyme into nearly anhydrous organic solvents. Interestingly, we report here that CYP2D6 colyophilized with trehalose and suspended in n-decane shows higher activity than in aqueous buffer. This study demonstrates the unexpected high tolerance of CYP2D6 to some low water organic solvents and provides an alternative strategy to facilitate the use of this enzyme in synthesis.     

Activity and batch operational stability of Candida rugosa lipase immobilized in different hydrophilic polyurethane foams during hydrolysis in a biphasic medium

The aim of this study was to investigate eventual relationships between some physico-chemical properties (e.g. porosity, aquaphilicity, partition coefficient for oleic acid and drying curves) of relatively hydrophilic polyurethane foams and the activity and batch operational stabiliy of Candida rugosa lipase immobilized within these foams. Two biocompatible polyurethane pre-polymers ("HYPOL FHP 2002^TM" and "Hypol FHP X4300^TM" from Hampshire Chemical GmbH, Germany) were tested as immobilization supports. The model reaction was the hydrolysis of crude olive residue oil in a biphasic aqueous/n-hexane medium. Drying curves under normal and reduced pressures suggested that water molecules are more strongly bound to the "FHP 2002" than to "FHP X4300" foams. This is in agreement with the higher aquaphilicity value estimated for the "FHP 2002" foam (3.7 vs 2.8). For every enzyme loading tested, hydrolysis efficiency was considerably higher for the lipase in "FHP X4300" foam when compared to the other counterpart. However, internal mass transfer limitations seem to be more severe with "FHP X4300" foams. Operational stability was evaluated in 10 consecutive batches (1 batch = 23 hours) for both immobilized preparations. A fast deactivation was observed for both biocatalysts. However, a slightly higher operational stability was observed for the lipase in "FHP 2002" foam. For the lipase in "FHP X4300" foam, the activity decay can be explained by a dramatic lipase leakage from the foam observed along successive batches. For the lipase in "FHP 2002" foam, no significant enzyme loss was observed along the reutilizations probably due to a higher number of multi-point attachment between the lipase and its support. In fact, activity and operational stability of Candida rugosa lipase in "FHP 2002" and "FHP X4300" foams appear to be related with the strength and/or the number of covalent binding between the enzyme and the support rather than to the physico-chemical properties evaluated in this work.