Subtilisin-catalysed peptide synthesis and transesterification in organic solvents

Summary Subtilisin from Bacillus subtilis was modified with polyethylene glycol (PEG), or adsorbed either on celite or porous glass, or directly used as a suspended powder to catalyse peptide synthesis and transesterification reactions in organic solvents. The rather low yield of peptide synthesis probably resulted from the enzyme tendency to catalyse hydrolysis and transesterification side reactions. The kinetics of transesterification catalysed by PEG-subtilisin was consistent with a ping-pong mechanism modified by a hydrolytic branch. Initial rates of transesterification were found to be dependent on alcohol and organic base concentrations in the reaction mixture. The high affinity of benzyloxycarbonyl-l-serine-methyl ester for the enzyme indicated that a change in substrate specificity of subtilisin occurred in organic phase. The 50-fold increase in the rate of synthesis of benzyloxycarbonyl-l-serine-l-phenylalanine amide which was observed when PEG-subtilisin was used instead of immobilized or powdered enzyme, suggested that a higher flexibility of the polypeptide chain modified by the covalent attachment of a number of soluble PEG moieties occurred in organic solvents. This also resulted in a lower stability of PEG-subtilisin at high temperature.Offprint requests to: A. Puigserver     

Optimization of Pseudomonas cepacia lipase preparations for catalysis in organic solvents

The activity of different lipase (from Pseudomonas cepacia) forms, such as crude powder (crude PC), purified and lyophilized with PEG (PEG + PC), covalently linked to PEG (PEG-PC), cross-linked enzyme crystals (CLEC-PC), and immobilized in Sol-Gel-AK (Sol-Gel-AK-PC) was determined, at various water activities (aw), in carbon tetrachloride, benzene and 1,4-dioxane. The reaction of vinyl butyrate with 1-octanol was employed as a model and both transesterification (formation of 1-octyl butyrate) and hydrolysis (formation of butyric acid from vinyl butyrate) rates were determined. Both rates depended on the lipase form, solvent employed, and aw value. Hydrolysis rates always increased as a function of aw, while the optimum of aw for transesterification depended on the enzyme form and nature of the solvent. At proper aw, some lipase forms such as PEG + PC, PEG-PC, and Sol-Gel-AK-PC had a total activity in organic solvents (transesterification plus hydrolysis) which was close to (39 and 48%) or even higher than (130%) that displayed by the same amount of lipase protein in the hydrolysis of tributyrin-one of the substrates most commonly used as standard for the assay of lipase activity-in aqueous buffer. Instead, CLEC-PC and crude PC were much less active in organic solvents (2 and 12%) than in buffer. The results suggest that enzyme dispersion and/or proper enzyme conformation (favored by interaction with PEG or the hydrophobic Sol-Gel-AK matrix) are essential for the expression of high lipase activity in organic media.     

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.     

Catalytic activity and conformation of chemically modified subtilisin Carlsberg in organic media

Subtilisin Carlsberg, an alkaline protease from Bacillus licheniformis, was modified with polyoxyethylene (PEG) or aerosol-OT (AOT), and the solubility, conformation, and catalytic activity of the modified subtilisins in some organic media were compared under the same conditions. The solubility of modified subtilisins depended on the solubility of the modifier. On the other hand, the conformational changes depended on the solubility, rather than the property, of the modifier. When the modified subtilisin was dissolved in water-miscible polar solvents such as dimethylsulfoxide, acetonitrile, and tetrahydrofuran, significant conformational changes occurred. When modified subtilisin was dissolved in water-immiscible organic solvents, such as isooctane and benzene, the solvent did not induce significant conformational changes. The catalytic activity in the transesterification reaction of the N-acetyl-L-phenylalanine ethylester of the modified subtilisin in organic solvents was higher than that of native subtilisin. The high activity of modified subtilisin was thought to be due to a homogeneous reaction by the dissolved enzymes.     

Obtaining high transesterification activity for subtilisin in ionic liquids

It is known that subtilisin shows poor transesterification activity in ionic liquids (ILs). The present work, taking subtilisin as the system, explores approaches for biocatalyst preparations, which are capable of yielding higher/adequate transesterification activity in these solvents. Of all the approaches tried, enzyme precipitated and rinsed with n-propanol (EPRP) gave the best results (about 10,000 times increase in initial rates in 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF(6)]) over what is obtained with pH tuned lyophilized powders). In case of water soluble ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim][BF(4)]), pH tuned lyophilized subtilisin did not show any transesterification activity. EPRP, however, gave an initial rate (for transesterification) of 2.78 mmol mg(-1) h(-1).     

Pentynyl dextran as a support matrix for immobilization of serine protease subtilisin Carlsberg and its use for transesterification of N-acetyl-l-phenylalanine ethyl ester in organic media

In this study, serine protease (subtilisin Carlsberg) was immobilized on pentynyl dextran (PyD, O–alkynyl ether of dextran, 1) and used for the transesterification of N-acetyl-l-phenylalanine ethyl ester (2) with different aliphatic (1-propanol, 1-butanol, 1-pentanol, 1-hexanol) and aromatic (benzyl alcohol, 2-phenyl ethanol, 4-phenyl-1-butanol) alcohols in tetrahydrofuran (THF). The effect of carbon chain length in aliphatic and aromatic alcohols on initial and average transesterification rate, transesterification activity of immobilized enzyme and yield of the reaction under selected reaction conditions was investigated. The transesterification reactivity of the enzyme and yield of the reaction increased as the chain length of the alcohols decreased. Furthermore, almost no change in yield was observed when the immobilized enzyme was repeatedly used for selected alcohols over six cycles. Intrinsic fluorescence analysis showed that the catalytic activity of the immobilized enzyme in THF was maintained due to retention of the tertiary structure of the enzyme after immobilization on PyD (1).     

Direct solubilization of enzyme aggregates with enhanced activity in nonaqueous media

A protein solubilization method has been developed to directly solubilize protein clusters into organic solvents containing small quantities of surfactant and trace amounts of water. Termed "direct solubilization," this technique was shown to solubilize three distinct proteins - subtilisin Carlsberg, lipase B from Candida antarctica, and soybean peroxidase - with much greater efficiencies than extraction of the protein from aqueous solution into surfactant-containing organic solvents (referred to as extraction). More significant, however, was the dramatic increase in directly solubilized enzyme activity relative to extracted enzyme activity, particularly for subtilisin and lipase in polar organic solvents. For example, in THF the initial rate towards bergenin transesterification was ca. 70 times higher for directly solubilized subtilisin than for the extracted enzyme. Furthermore, unlike their extracted counterparts, the directly solubilized enzymes yielded high product conversions across a spectrum of non-polar and polar solvents. Structural characterization of the solubilized enzymes via light scattering and atomic force microscopy revealed soluble proteins consisting of active enzyme aggregates containing approximately 60 and 100 protein molecules, respectively, for subtilisin and lipase. Formation of such clusters appears to provide a microenvironment conducive to catalysis and, in polar organic solvents at least, may protect the enzyme from solvent-induced inactivation.     

Incorporation of a stabilizing Ca(2+)-binding loop into subtilisin BPN'.

A rational approach was taken to improve the stability of subtilisin BPN' to autoproteolysis. Two sites of autoproteolysis were identified by isolation of early autolysis products and amino-terminal sequence analysis. These studies showed that subtilisin rapidly cleaves Ala48-Ser49 and Ser163-Thr164 peptide bonds at elevated temperatures. These two sites appear in regions of high mobility as estimated from crystallographic B-factors and are in extended surface loops. To improve the resistance to thermal-induced autolysis, we replaced sequences around these two sites with sequences derived from a thermophilic homologue of subtilisin, thermitase. Thermitase contains a Ca(2+)-binding site in the region surrounding Ser49. When the Ca(2+)-binding segment of thermitase corresponding to residues 45-63 of subtilisin BPN' was installed into subtilisin BPN', the chimeric protein gained the ability to bind another Ca2+ with moderate affinity (Kd approximately 100 microM). This enzyme had the same kcat as wild-type, had a KM value 8-fold larger than wild-type, and was slightly less stable to thermal inactivation in EDTA. However, in 10 mM CaCl2, the mutant subtilisin BPN' was 10-fold more stable to irreversible inactivation at 60 degrees C than wild-type subtilisin BPN' as measured by residual activity against the substrate sAAPF-pna. Next, mutations and deletions derived from thermitase were introduced near the second autolysis loop in subtilisin BPN' (residues 158-165). However, all of these mutants were less stable than wild-type subtilisin. Thus, some (but not all) mutations derived from a thermophilic homologue near sites of autolysis can be stabilizing to a mesophilic protease.     

A lipid-coated lipase as an efficient hydrolytic catalyst in the two-phase aqueous-organic system

The activity of different formulations of Candida antarctica lipase B (CALB), such as crude CALB, purified CALB, purified CALB lyophilized with PEG (CALB + PEG) or oleic acid (CALB + OA), and the commercial formulation Novozym 435, was determined in toluene, carbon tetrachloride, and 1,4-dioxane at various water activities (a(w)). The reaction between vinylacetate and 1-octanol was used as the model reaction and both transesterification (formation of 1-octylacetate) and hydrolytic (formation of acetic acid from vinylacetate) activities were determined. For equal amounts of lipase protein, CALB + PEG (and to a lesser extent CALB + OA) displayed higher activity than that of the other formulations; for instance, in toluene (a(w) < 0.1), it was 260-, 13-, and 1.8-fold more active than crude CALB, purified CALB, and Novozym 435, respectively. Moreover, the transesterification activity of CALB + PEG was of the same order of magnitude (51%) of the activity shown by the enzyme in the hydrolysis of vinylacetate in aqueous buffer. These results suggest that PEG and oleic acid could act as lyoprotectants, preventing the formation of intermolecular interactions during the lyophilization process that might be responsible for protein denaturation. No diffusional limitation was observed for CALB + PEG-catalyzed reactions. Purified CALB, in contrast to the other formulations, showed a marked activity increase (2.1 to 7.8-fold) as a function of a(w) and, in 1,4-dioxane, it was 3.5-fold more active when it was added to the solvent after previous dissolution of the lyophilized powder in water.     

Solvent effects on the catalytic activity of subtilisin suspended in compressed gases

We studied a model transesterification reaction catalyzed by subtilisin Carlsberg suspended in carbon dioxide, propane, and mixtures of these solvents under pressure. To account for solvent effects due to differences in water partitioning between the enzyme and the bulk solvents, we measured water sorption isotherms for the enzyme in each solvent. We measured catalytic activity as a function of enzyme hydration and obtained bell-shaped curves with maxima at the same enzyme hydration (12%) in all the solvents. However, the activity maxima were different in all media, being much higher in propane than in either CO(2) or the mixtures with 50 and 10% CO(2). Considerations based on the solvation ability of the solvents did not offer an explanation for the differences in catalytic activity observed. Our results suggest that CO(2) has a direct adverse effect on the catalytic activity of subtilisin. (c) 1996 John Wiley & Sons, Inc.     

Correlation between catalytic activity and secondary structure of subtilisin dissolved in organic solvents

Fourier-transform infrared (FTIR) spectroscopy has been used to quantify the alpha-helix and beta-sheet contents of subtilisin Carlsberg dissolved in several nonaqueous, as well as aqueous, solvents. Independently, the catalytic activity of the enzyme has been measured in the same solvents. While our previous FTIR studies revealed no connection between the secondary structure and enzymatic activity for subtilisin suspended in various organic solvents, a very different situation is observed herein for the dissolved enzyme. Specifically, if either the alpha-helix or beta-sheet content in a given solvent is higher or lower than in water, no appreciable enzymatic catalysis is observed. Conversely, when the secondary structure of subtilisin dissolved in a given nonaqueous solvent is similar to that in water, so is the enzymatic activity. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 485-491, 1997.     

Comparison of theoretical and experimental data to evaluate substrate diffusional limitations for crown ether- and methyl-beta-cyclodextrin-activated serine protease subtilisin Carlsberg in tetrahydrofuran

Simple co-lyophilization of serine protease subtilisin Carlsberg with [12]-crown ether-4 (12-crown-4) or methyl-beta-cyclodextrin (MbetaCD) drastically increases its catalytic activity in organic solvents. We investigated whether the improved activity would cause substrate diffusional limitations. To experimentally assess the issue, the enzyme was inactivated with PMSF. Different amounts of active and inactive subtilisin were codissolved in 10 mM phosphate buffer (pH 7.8) followed by lyophilization with or without 12-crown-4 or MbetaCD. Initial rates for the transesterification reaction of N-acetyl-L-phenylalanine ethyl ester and 1-propanol in anhydrous THF were plotted vs. the amount of active enzyme present in the formulations. For all three enzyme formulations a linear relationship was observed and the results clearly show that activation of subtilisin Carlsberg by crown ethers and MbetaCD did not cause diffusional limitations. This was somewhat surprising because theoretical models predicted such diffusional limitations for the activated formulations. However, investigation of the protein powder particles obtained after co-lyophilization with 12-crown-4 and MbetaCD revealed a drastically reduced particle size for these formulations when suspended in THF. The particle micronization afforded by the excipients prevented substrate diffusional limitations, a factor that should be taken into account when designing improved enzyme formulations for synthetic applications in organic solvents.     

Thiol-functionalization of acrylic ester monomers catalyzed by immobilized Humicola insolens cutinase

Immobilized cutinase HiC from the ascomycete Humicola insolens was applied as a novel biocatalyst for the synthesis of functionalized acryclic esters by transesterification. As a model reaction, transesterification of methyl acrylate with 6-mercapto-1-hexanol at a high molar ratio in a solvent free system was chosen. Besides two minor Michael-addition by-products, 6-mercaptohexyl acrylic ester was identified as the main product with the thiol as the functional end group. Reaction conditions were optimized regarding the influence of water (0-1.72 M), temperature (22-50 °C), product inhibition and addition of the radical inhibitor butylated hydroxytoluol (BHT; 0.14-0.71 M) on conversion and by-product formation. Highest conversion of 6-mercapto-1-hexanol to 6-mercaptohexyl acrylic ester (95.4 ± 0.3%) was achieved after 6h at 40 °C in the presence of 0.025% (w/w) water without formation of by-products in a solvent free system. Applying methyl methacrylate, transesterification with 6-mercapto-1-hexanol was significantly lower (43.6 ± 0.1%) compared to transesterification of methyl acrylate with 6-mercapto-1-hexanol.     

Enzyme activation in organic solvents: co-lyophilization of subtilisin Carlsberg with methyl-beta-cyclodextrin renders an enzyme catalyst more active than the cross-linked enzyme crystals

In this study we explored the efficiency of the additive methyl-beta-cyclodextrin (M beta CD) to enhance the activity and enantioselectivity of the serine protease subtilisin Carlsberg in organic solvents. These two parameters, measured for different transesterification reactions and in several solvents, are compared with results obtained by using two additional preparations of the same enzyme: lyophilized powder and cross-linked enzyme crystals (CLEC). The results suggest that co-lyophilization of subtilisin with M beta CD preserves the enzyme's active site tertiary structure rendering a highly active and enantioselective catalyst.     

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.     

Protease activation in glycerol-based deep eutectic solvents

Deep eutectic solvents (DESs) consisting of mixtures of a choline salt (chloride or acetate form) and glycerol are prepared as easily accessible, biodegradable, and inexpensive alternatives to conventional aprotic cation-anion paired ionic liquids. These DES systems display excellent fluidity coupled with thermal stability to nearly 200 °C. In this work, the transesterification activities of cross-linked proteases (subtilisin and α-chymotrypsin), immobilized on chitosan, were individually examined in these novel DESs. In the 1:2 molar ratio mixture of choline chloride/glycerol containing 3% (v/v) water, cross-linked subtilisin exhibited an excellent activity (2.9 μmo l min(-1) g(-1)) in conjunction with a selectivity of 98% in the transesterification reaction of N-acetyl-L-phenylalanine ethyl ester with 1-propanol. These highly encouraging results advocate more extensive exploration of DESs in protease-mediated biotransformations of additional polar substrates and use of DESs in biocatalysis more generally.     

Factors limiting the hydrolysis of casein by subtilisin DY

It was shown that during the subtilisin DY-induced hydrolysis of casein relatively stable polypeptide structures are formed. In their interior these structures contain peptide bonds which are susceptible to the enzyme used. Heating (up to 100 degrees C) and/or application of ultrasound (25 kHz, 60 W) results in their unfolding. Data are provided, which show that under the enzyme-substrate complex formation does not lead to an enzyme conformation more susceptible to autolysis. Taking into account the described phenomena a higher degree of hydrolysis was attained in comparison to those obtained by standard enzymatic hydrolysis.     

Remarkable activation of enzymes in nonaqueous media by denaturing organic cosolvents

The rates of transesterification reactions catalyzed by the protease subtilisin Carlsberg suspended in various anhydrous solvents at 30 degrees C can be increased more than 100-fold by the addition of denaturing organic cosolvents (dimethyl sulfoxide or formamide); in water, the same cosolvents exert no enzyme activation. At 4 degrees C, the activation effect on the lyophilized protease is even higher, reaching 1000-fold. Marked enhancement of enzymatic activity in anhydrous solvents by formamide is also observed for two other enzymes, alpha-chymotrypsin and Rhizomucor miehei lipase, and is manifested in two transesterification reactions. In addition to lyophilized subtilisin, crosslinked crystals of subtilisin are also amenable to the dramatic activation by the denaturing cosolvents. In contrast, subtilisin solubilized in anhydrous media by covalent modification with poly(ethylene glycol) exhibits only modest activation. These observations are rationalized in terms of a mechanistic hypothesis based on an enhanced protein flexibility in anhydrous millieu brought about by the denaturing organic cosolvents. The latter exert their lubricating effect largely at the interfaces between enzyme molecules in a solid preparation, thus easing the flexibility constraints imposed by protein-protein contacts. (c) 1996 John Wiley & Sons, Inc.     

Enhancement of the thermostability of subtilisin E by introduction of a disulfide bond engineered on the basis of structural comparison with a thermophilic serine protease

Sites for Cys substitutions to form a disulfide bond were chosen in subtilisin E from Bacillus subtilis, a cysteine-free bacterial serine protease, based on the structure of aqualysin I of Thermus aquaticus YT-1 (a thermophilic subtilisin-type protease containing two disulfide bonds). Cys residues were introduced at positions 61 (wild-type, Gly) and 98 (Ser) in subtilisin E by site-directed mutagenesis. The Cys-61/Cys-98 mutant subtilisin appeared to form a disulfide bond spontaneously in the expression system used and showed a catalytic efficiency equivalent to that of the wild-type enzyme for hydrolysis of a synthetic peptide substrate. The thermodynamic characteristics of these enzymes were examined in terms of enzyme autolysis (t1/2) and thermal stability (Tm). The half-life of the Cys-61/Cys-98 mutant was found to be 2-3 times longer than that of the wild-type enzyme. Similar results were obtained by differential scanning calorimetry. The disulfide mutant showed a Tm of 63.0 degrees C, which was 4.5 degrees C higher than that observed for the wild-type enzyme. Under reducing conditions, however, the characteristics of the mutant enzyme were found to revert to those of the wild-type enzyme. These results strongly suggest that the introduction of a disulfide bond by site-directed mutagenesis enhanced the thermostability of subtilisin E without changing the catalytic efficiency of the enzyme.     

A comparison of lipase-catalysed ester and lactone synthesis in low-water systems: analysis of optimum water activity.

We investigated the effects of the lyophilisation medium (enzyme plus buffer salt and additives) and of water activity (a(w)) on the catalytic properties of lipase from Chromobacterium viscosum (lipase CV) in organic solvents; catalysis of ester and lactone synthesis were compared and, despite the similarities of the reactive groups involved in these reactions, some interesting differences were observed. Including 2-[N-morpholino]ethanesulfonic acid (MES) buffer in the lyophilisation medium of lipase CV increased its catalytic activity in transesterification and lactonisation, although the buffer salt requirement for maximal activity differed between the two reactions. Sorbitol, glucose, lactose, 18-crown-6 (crown ether 18-C-6), beta-cyclodextrin and bovine serum albumin were employed as alternative additives in the transesterification reaction, but were not as effective as MES buffer. Salt hydrates were used to investigate the effect of a(w) on esterification and lactonisation reactions catalysed by lipase CV. The maximum rate of hexadecanolide synthesis in toluene occurred at a(w) = 0.48. The optimum a(w) for the transesterification reaction in heptane/alcohol mixtures depended on the alcohol substrate employed (1-heptanol, 2-heptanol, or 3-methyl-3-hexanol) but not on the acyl donor (p-NP acetate or caprylate). The optimum a(w) values for both reactions were unchanged when a common solvent system (toluene/1-heptanol) was employed, indicating that the dependence of enzyme activity on a(w) is an intrinsic property of the enzyme-catalysed reaction and not a function of the solvent or other additives.