Native and modified subtilisin Karlsberg as an effective catalyst of peptide bond formation in organic media

The activity and stability of native subtilisin Karlsberg and subtilisin 72 and their complexes with sodium dodecyl sulfate (SDS) in organic solvents were studied. The kinetic constants of the hydrolysis of specific chromogenic peptide substrates Z- ALA-Ala-Leu-pNA and Glp-Ala-Ala-Leu-pNA by the subtilisins were determined. It was found that the subtilisin Karlsberg complex with SDS in anhydrous organic solvents is an effective catalyst of peptide synthesis with multifunctional amino acids in positions P1 and P'1 (Glu, Arg, and Asp) containing unprotected side ionogenic groups.     

Modified proteinases in peptide synthesis in organic media

We showed that modified proteases could catalyze synthesis of a wide variety of peptides of various lengths and structures both in solution and on solid phase in organic solvents. The following modified proteases were studied as catalysts for enzymatic peptide synthesis in polar organic solvents (acetonitrile, dimethylformamide, and ethanol): pepsin sorbed on celite, a noncovalent complex of subtilisin with sodium dodecylsulfate, and subtilisin or thermolysin covalently immobilized on a cryogel of polyvinyl alcohol. The use of the noncovalent complex of subtilisin with sodium dodecylsulfate and immobilized subtilisin is especially promising for the segment condensation of peptide fragments containing residues of trifunctional amino acids with unprotected ionogenic groups in side chains, such as Lys, Arg, His, Glu, and Asp.     

Modified Proteases for Peptide Synthesis in Organic Media

We showed that modified proteases could catalyze synthesis of a wide variety of peptides of various lengths and structures both in solution and on solid phase in organic solvents. The following modified proteases were studied as catalysts for enzymatic peptide synthesis in polar organic solvents (acetonitrile, dimethylformamide, and ethanol): pepsin sorbed on celite, a noncovalent complex of subtilisin with sodium dodecylsulfate, and subtilisin or thermolysin covalently immobilized on a cryogel of polyvinyl alcohol. The use of the noncovalent complex of subtilisin with sodium dodecylsulfate and immobilized subtilisin is especially promising for the segment condensation of peptide fragments containing residues of trifunctional amino acids with unprotected ionogenic groups in side chains, such as Lys, Arg, His, Glu, and Asp.     

Native subtilisin Karlsberg and modified subtilisin 72 as effective catalysts of peptide bond formation in organic media

The activity and stability of native subtilisin Karlsberg and subtilisin 72 and their complexes with sodium dodecyl sulfate (SDS) in organic solvents were studied. The kinetic constants of the hydrolysis of specific chromogenic peptide substrates Z-Ala-Ala-Leu-pNA and Glp-Ala-Ala-Leu-pNA by the subtilisins were determined. It was found that the subtilisin Karlsberg complex with SDS in anhydrous organic solvents is an effective catalyst of peptide synthesis with multifunctional amino acids in positions P ₁ and P ₁ ^′ (Glu, Arg, and Asp) containing unprotected side ionogenic groups.     

Enzymatic reactions in aqueous-organic media. VIII. Medium effect and nucleophile specificity in subtilisin-catalyzed peptide synthesis in organic solvents

Summary Subtilisin Carlsberg and subtilisin BPN' (nagarse) catalyze peptide bond formation from aromatic amino acid esters and glycinamide in hydrophilic organic solvents. The activities of subtilisin and product compositions are different in several organic solvents; reactions in acetonitrile, tetrahydrofuran, and propylene carbonate gave the peptide in excellent yields, while in N,N-dimethylformamide and methanol the enzyme activity was largely retarded. The yield of the peptide is also dependent on water content in the reaction solutions. Optimum water contents are in the range from 3 to 7 %. The reaction is strongly specific for glycinamide as an amine component, and amides of alanine, valine, and leucine gave the corresponding peptides in poor yields.     

Native and modified subtilisin 72 as a catalyst of peptide synthesis in media with low water content

The catalytic efficiencies of native subtilisin, its noncovalent complex with polyacrylic acid, and the subtilisin covalently immobilized in a cryogel of polyvinyl alcohol were studied in the reaction of peptide coupling in mixtures of organic solvents with a low water content in dependence on the medium composition, reaction time, and biocatalyst concentration. It was established that, in media with a DMF content > 80%, the synthase activity of modified subtilisins is higher than that of the native subtilisin. The use of N-acylpeptides with a free carboxyl group was found to be possible in organic solvents during the enzymatic synthesis catalyzed by both native and immobilized subtilisin. A series of tetrapeptide p-nitroanilides of the general formula Z-Ala-Ala-Xaa-Yaa-pNA (where Xaa is Leu, or Glu and Yaa is Phe or Asp) was obtained in the presence of immobilized enzyme in yields of 70-98% in DMF-MeCN without any activation of the carboxyl component and without protection of side ionogenic groups of polyfunctional amino acids.     

The applicability of subtilisin Carlsberg in peptide synthesis.

The synthesis of peptide bonds catalysed by subtilisin Carlsberg was studied in different hydrophilic organic solvents with variable H2O concentration. Z-Val-Trp-OMe and Z-Ala-Phe-OMe were used as acyl donors, and a series of amino acid derivatives, di- and tripeptides of the general structure Xaa-Gly, Gly-Xaa, Gly-Gly-Xaa (Xaa represents all natural L-amino acids except cysteine) and other peptides were used as nucleophiles. A comparative study of the enzymatic synthesis in aqueous DMF (50%, v/v) and acetonitrile containing 10% (v/v) of H2O demonstrated that the yields of peptide products were higher in most cases when acetonitrile with low H2O concentration was used. The acylation of weak nucleophiles was improved in organic solvents with very low H2O concentration (2%). The reactions in anhydrous Bu(t)-OH proceeded with substantially lower velocity. Generally, the restricted nucleophile specificity of the enzyme for glycine and hydrophilic amino acid residues in P1' position, as well as numerous side reactions, limit the utilization of subtilisin in peptide synthesis, especially in the case of the segment condensations. Contrary to the published data, we have proved that proline derivatives were not acylated in any media with the help of subtilisin Carlsberg. Effective ester hydrolysis of a protected nonapeptide corresponding to the N-terminal sequence of dicarba-eel-calcitonin catalysed by subtilisin was achieved.     

Peptide synthesis by proteases in organic solvents: medium effect on substrate specificity.

The substrate specificities of alpha-chymotrypsin and subtilisins for peptide synthesis in hydrophilic organic solvents were investigated. Chymotrypsin exhibited high specificity to aromatic amino acids as acyl donors, while subtilisin Carlsberg and subtilisin BPN' were specific to aromatic and neutral aliphatic amino acids, in accordance with the S1 specificities of the enzymes for peptide hydrolysis in aqueous solutions. On the contrary, chymotrypsin exhibited higher specificities to hydrophilic amino acid amides as acyl acceptors (nucleophiles) for peptide synthesis with N-acetyl-L-tyrosine ethyl ester, in contrast to the S1' specificity for peptide hydrolysis and peptide synthesis in aqueous solutions. Furthermore, nucleophile specificity changed with the change in water-organic solvent composition; the increase in water content led to increase in relative reactivity of leucinamide to that of alaninamide. It was also found that protection of the carboxyl group of alanine by amidation is much preferable to protection by esterification in terms of reactivity as nucleophiles.     

Native and Modified Subtilisin 72 as a Catalyst for Peptide Synthesis in Media with a Low Water Content

The catalytic efficiencies of native subtilisin, its noncovalent complex with polyacrylic acid, and the subtilisin covalently immobilized in a cryogel of polyvinyl alcohol were studied in the reaction of peptide coupling in mixtures of organic solvents with a low water content in dependence on the medium composition, reaction time, and biocatalyst concentration. It was established that, in media with a DMF content >80%, the synthase activity of modified subtilisins is higher than that of the native subtilisin. The use of N-acylpeptides with a free carboxyl group was found to be possible in organic solvents during the enzymatic synthesis catalyzed by both native and immobilized subtilisin. A series of tetrapeptide p-nitroanilides of the general formula Z-Ala-Ala-Xaa-Yaa-pNA (where Xaa is Leu, Lys, or Glu and Yaa is Phe or Asp) was obtained in the presence of immobilized enzyme in yields of 70–98% in DMF–MeCN without any activation of the carboxyl component and without protection of side ionogenic groups of polyfunctional amino acids.     

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.     

Peptide synthesis using proteases dissolved in organic solvents

Organic solvent-soluble -chymotrypsin (CT) and subtilisin Carlsberg (SC) are effective catalysts for peptide synthesis in homogeneous organic solutions. The soluble enzymes have values of k_cat/K_m for the reaction of N-Bz-L-Tyr-OEt with L-Leu-NH₂ to yield the dipeptide N-Bz-L-Tyr-L-Leu-NH₂ that are over 3 orders of magnitude higher than their suspended counterparts in isooctane (containing 30% (v/v) tetrahydrofuran (THF) to aid in substrate solubility). Both enzymes are substantially more active in hydrophobic organic solvents than hydrophilic solvents. Adding small concentrations of water (<0.2% and 1% (v/v) in isooctane-THF and ethyl acetate, respectively) results in up to a 150-fold activation of -chymotrypsin-catalyzed peptide synthesis. Importantly, added water does not promote hydrolysis in either isooctane-THF or ethyl acetate; thus, -chymotrypsin is highly selective toward peptide synthesis in the nearly anhydrous organic solutions. Unlike CT, the activation of subtilisin Carlsberg upon partial hydration of isooctane-THF or ethyl acetate was not significant and actually resulted in substantial hydrolysis. Using -chymotrypsin, a variety of tripeptides were produced from dipeptide amino acid esters. Reactivity of D-amino acid amides as acyl acceptors and partially unblocked amino acid acyl donors further expands the generality of the use of organic solvent-soluble enzymes as peptide synthesis catalysts.     

The Effects of Organic Solvents on Wild-Type and Mutant Subtilisin-Catalyzed Hydrolyses

The kinetic parameters, k_cat and K_M, for the hydrolysis of N-α-tosyl-L-arginine methyl ester (1, TAME) by the wild-type subtilisins Carlsberg and BPN′ as well as the BPN′ mutants Glyl66Ser, GLyl66Asn, and Met222Phe, were determined in the presence of 5 and 15% (v/v) of a selection of water-soluble organic solvents. The goals were to compare and evaluate the solvent effects with a view to expanding their use in organic synthetic applications of the WT and mutant subtilisins. The results showed that subtilisin BPN′ and its mutants were much less affected by organic solvents than subtilisin Carlsberg. The BPN′ mutant Met222Phe demonstrated the greatest resistance to cosolvent inactivation, making it a particularly attractive mutant for peptide synthesis. Dimethyl sulfoxide, acetone, and branched alcohols were found to be the most benign solvents, whereas dioxane, THF, and N-methyl-2-pyrrolidinone seriously reduced catalytic activities, even at low concentrations. The results parallel the solvent-effect data available for other proteinases, including α-chymotrypsin.     

Immobilization of proteases to porous chitosan beads and their catalysis for ester and peptide synthesis in organic solvents.

alpha-Chymotrypsin (CT), subtilisin BPN' (STB), and subtilisin Carlsberg (STC) were immobilized by adsorption to porous chitosan beads (Chitopearl, CP). The immobilized enzymes showed higher catalytic activities than free enzymes for amino acid esterification in many hydrophilic organic solvents except for methanol and DMF. In ethanol, the initial rate of the esterification increased with water content, whereas in ethyl acetate, the maximum rate was obtained at 2%-3% water. CP-immobilized CT also catalysed transesterification of Ac-Tyr-OMe in ethanol and peptide synthesis in acetonitrile from Ac-Tyr-OH or its ethyl ester and amino acid amides. The immobilized enzymes are highly stable in organic solutions, and can easily be separated from the reaction solutions. Repeated esterifications of Ac-Tyr-OH in acetonitrile by a CP-immobilized CT gave almost constant yields of the ester for more than 3 weeks.     

Influence of solvent and water activity on kinetically controlled peptide synthesis.

alpha-Chymotrypsin deposited on Celite was used to catalyse peptide synthesis reactions between N-protected amino acid esters and leucine amide in organic media with low water content. The influence of the solvent and the thermodynamic water activity on the reaction kinetics was studied. The substrate specificity in the reactions was shown to be a combination of the substrate specificity of the enzyme in aqueous media and the influence of the solvents. The magnitude of the solvent effects differed greatly depending on the substrates used. In hydrophobic solvents high reaction rates were observed and the competing hydrolysis of the ester substrate occurred to only a minor extent. Reactions occurred at water activities as low as 0.11, but the rate constants increased with increasing water activity and were about two orders of magnitude higher at the highest water activity tested (0.97).     

Synthesis of peptide aldehydes via enzymatic acylation of amino aldehyde derivatives

Two ways for semi-enzymatic preparation of the peptide aldehydes are proposed: (1) enzymatic acylation of amino alcohols with acyl peptide esters and subsequent chemical oxidation of the resulting peptide alcohols with DMSO/acetic anhydride mixture or (2) enzymatic acylation of the preliminarily obtained by a chemical route amino aldehyde semicarbazones. Subtilisin 72, serine proteinase with a broad specificity, distributed over macroporous silica, was used as a catalyst in both cases. Due to the practical absence of water in the reaction mixtures the yields of the products in both enzymatic reactions were nearly quantitative. The second way seems to be more attractive because all chemical stages were carried out with amino acid derivatives, far less valuable compounds than peptide ones. A series of peptide aldehydes of general formula Z-Ala-Ala-Xaa-al (where Xaa-al=leucinal, phenylalaninal, alaninal, valinal) was obtained. The inhibition parameters for these compounds, in the hydrolysis reactions of corresponding chromogenic substrates for subtilisin and -chymotrypsin, were determined.     

Chemoselective protection of the amino groups of chitosan by controlled phthaloylation: facile preparation of a precursor useful for chemical modifications

A simple and convenient procedure for chemoselectively protecting the amino groups of chitosan has been developed to provide N-phthaloyl-chitosan that is indispensable as a soluble N-protected precursor for further controlled modification reactions of chitosan. Although the conventional N-phthaloylation of chitosan in N,N-dimethylformamide was accompanied by partial phthaloylation of the hydroxy groups, the addition of a small amount of hydroxy-containing compounds effectively suppressed the O-phthaloylation. Of some compounds examined, water proved particularly suitable, resulting in the formation of chemoselectively N-phthaloylated chitosan without any appreciable O-phthaloyl groups. The resulting N-phthaloyl-chitosan was found to be crystalline despite the presence of a bulky substituent. A solubility test indicated that N-phthaloyl-chitosan exhibited considerable affinity for organic solvents.     

Enzymatic synthesis of amino acid-sugar alcohol conjugates in organic media

Amino acid-sugar alcohol conjugates were synthesized by a commercial serine protease, Optimase M-440, in organic media. Optimase M-440 showed broad substrate specificity towards N-t-Boc-protected l-amino acids as acyl donors and sugar alcohols as nucleophiles. Among various solvents tested Optimase M-440 showed the highest activity in pyridine. The regioselective acylation of the primary –OH groups of sugar alcohols gave the amino acid conjugates in good yields without byproducts.     

The role of conformational flexibility of enzymes in the discrimination between amino acid and ester substrates for the subtilisin-catalyzed reaction in organic solvents

To investigate how the conformational flexibility of subtilisin affects its ability to discriminate between enantiomeric amino acid and ester substrates for the subtilisin-catalyzed reaction in an organic solvent, the flexibility around the active site and the surface of subtilisin was estimated from the mobility of a spin label bound to subtilisin by ESR spectroscopy. Many studies on enzyme flexibility focus on the active site. Both the surface and active site flexibility play an important role in the enantioselectivity enhancement of the enzyme-catalyzed reaction. It was found, however, that the different behavior observed for the enantioselectivity between the amino acid and ester substrates could be correlated with the flexibility around the surface rather than the flexibility at the active site of subtilisin. In other words, for the ester substrates, the greater flexibility around the surface of subtilisin induced by a conformational change resulting from the presence of an additive such as DMSO is essential for the enantioselectivity enhancement. This model is also supported by the Michaelis-Menten kinetic parameters for each enantiomeric substrate. Our findings provide insight into the enantioselectivity enhancement for the resolution of enantiomers for enzyme-catalyzed reactions in organic solvents.     

Enzyme engineering for nonaqueous solvents. II. Additive effects of mutations on the stability and activity of subtilisin E in polar organic media.

Single amino acid substitutions increase the activity and stability of subtilisin E in mixtures of organic solvents and water, and the effects of these mutations are additive. A variant of subtilisin E that exhibits higher activity in mixtures of dimethylformamide (DMF) and water (Q103R) was created by random mutagenesis combined with screening for improved activity (K. Chen and F. H. Arnold, in preparation). Another mutation, N218S, known to improve both the activity and stability of subtilisin BPN', also improves the activity and stability of subtilisin E in the presence of DMF. The effects of the two substitutions on transition-state stabilization are additive. Furthermore, the Q103R mutation that improves activity has no deleterious effect on subtilisin stability. The double mutant Q103R+N218S is 10 times more active than the wild-type enzyme in 20% (v/v) DMF and twice as stable in 40% DMF. Although the effects of single mutations can be impressive, a practical strategy for engineering enzymes that function in nonaqueous solvents will most likely require multiple changes in the amino acid sequence. These results demonstrate the excellent potential for engineering nonaqueous-solvent-compatible enzymes.     

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.