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.     

The influence of bond chemistry on immobilized enzyme systems for ex vivo use

The ideal derivatized support for the clinical use of an immobilized enzyme system should irreversibly bind active enzyme. We have investigated the behavior of heparinase and bilirubin oxidase immobilized via cyanogen bromide, tresyl chloride, epoxide, or carbodiimidazole activated natural and synthetic matrices. The protein bound to each activated support was 90% for cyanogen bromide (CNBr) activated agarose, 50-80% for tresyl chloride activated agarose, and 50% for oxirane activated acrylic (Eupergit C). The activity retention of immobilized heparinase was greatest (50%) with CNBr activated agarose while for the immobilization of bilirubin oxidase, the activity retention was greatest (25-30%) with tresyl chloride activated agarose and oxirane activated acrylic.The stability of the different covalent bonds was studied in vitro with radioiodinated enzymes. The leaching profiles showed the same trends for each support and chemistry. A plateau in portein leaching was reached after a few hours of incubatttion and the transient leaching period was well represented byu a logarithimic function of time. The amount of enzyme released from the least stable support (CNBr activated agarose) in 24 h was injected intravenously in New Zealand white rabbits. Using an indirect enzyme-linked immunnosorbant assay (ELISA), no immune responce was detected. The transient leaching profile was shortenend by washingthe enzyme-support conjugate with 1M hydroxylamine, pH8.5 intermolecular cross-linking with glutaraldehyde also improves the enzyme-support stability. Tresyl chloride and oxirane activated supports produce bonds with improved stability without adversely affecting enzymatic activity.     

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.     

Peptide synthesis with a proteinase from the extremely thermophilic organism Thermus Rt41A

A proteinase isolated from Thermus RT41a was immobilized to controlled pore glass beads and was used in the free and immobilized forms for peptide synthesis. The observed maximum yield was the same in both cases. a number of dipeptides were produced from amino acid esters and amides. The best acyl components, from those tested, were found to be Ac-Phe-OEt and Bz-Ala-OMe. Tur-NH(2), Trp-NH(2), Leu-pNA, and Val-pNA were all reactive nucleophiles.The kinetically controlled synthesis of Bz-ala-Tyr-NH(2) was optimized by studying the effect of pH, temperature, solvent concentration, ionic strength, and nucleophile and acyl donor concentration, ionic strength, and nucleophile and acyl donor concentration on the maximum yield. The initial conditions used were 25 mM Bz-ala-OMe, 25 mM Tyr-NH(2), 70 degrees C, pH 8.0, and 10% v/v dimethylformamide (DMF). The optimum conditions were 90% v/v DMF using 80 mM bz-Ala-OMe and 615 mM Tyr-NH(2) at 40 degrees C and pH 10. These conditions increased the maximum conversion from 0.75% to 26% (of the original ester concentration). In a number of other cosolvents, the best peptide yields were observed with acetonitrile and ethyl acetate. In 90% acetonitrile similar yields were observed to those in 90% DMF under optimized conditions except that the acyl donor and nucleophile concentrations could be reduced to 25 mM and 100mM, respectively. The effect of the blocking group on the nucleophile was also investigated; -betaNA and -pNA as blocking groups improved the yields markedly. The blocking and leaving groups of the acyldonor had no effect on the dipeptide yield. (c) 1994 John Wiley & Sons, Inc.     

Biocatalytic properties of native and immobilized subtilisin 72 in aqueous-organic and low water media

Subtilisin 72 was immobilized on cryogel of poly(vinyl alcohol), the macroporous carrier prepared by the freeze-thaw-treatment of concentrated aqueous solution of the polymer. The obtained biocatalyst was active and stable in aqueous, aqueous-organic, as well as in low water media. The stability of immobilized biocatalyst was substantially higher than that of native enzyme in all mixtures especially in aqueous buffer containing 5–8 M Urea and in acetonitrile/60–90%DMF mixtures. The ability of native and immobilized subtilisin to catalyze peptide bond formation between Z-Ala-Ala-Leu-OMe and Phe-pNA was studied in non-aqueous media. Considerable enzyme stabilization in acetonitrile/90%DMF mixture, induced by the immobilization, resulted in higher product yield (57%) than in case of native subtilisin suspension (32%). Detailed study of synthesis reaction revealed that notable increase in product yield could be reached using increase in both substrate concentrations up to 200 mM.     

Carboxypeptidase Y-Catalyzed Reaction in Systems Containing Organic Solvent

The effect of organic solvents on carboxypeptidase Y (a serine carboxypeptidase from yeast)-catalyzed hydrolysis of amino acid ester and peptide synthesis from N-acyl amino acid ester and amino acid amide was investigated.

The K_m value of ester hydrolysis increased with an increase in the solvent content. Dioxane was the most effective and dimethyl sulfoxide (DMSO) the least, whilst K_cat showed a tendency to increase slightly in N, N-dimethylformamide (DMF) and DMSO. For dioxane and acetonitrile (MeCN) a maximum was observed.

In peptide formation from Fua-Phe-OEt and Gly-NH₂, dioxane and MeCN supported high product yield at molar fractions smaller than ca. 0.05 but the yield decreased significantly at higher fractions, although a relatively constant selectivity (ratio of the peptide bond formed to the ester consumed) was maintained. DMSO gave rather low peptide yields and selectivity even at lower molar fractions. DMF showed an intermediate tendency.

An apparent saturation parameter of the amine component was evaluated and the dissociation constant of a complex between acyl-enzyme and amino acid amide (K_n), as well as the rate constant of aminolysis exerted by the amino acid amide bound correctly on the enzyme (K_n), was calculated by initial rate analysis of peptide formation. In contrast to K_m values, K_n decreased with increasing concentrations of organic cosolvent. while a suppressive effect was observed (except for DMSO) on the K_n parameter.

Effects of the solvent practically immiscible in water was also studied by use of the enzyme physically “immobilized” on glass beads.     

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.     

Carboxypeptidase Y-Catalyzed Reaction in Systems Containing Organic Solvent

The effect of organic solvents on carboxypeptidase Y (a serine carboxypeptidase from yeast)-catalyzed hydrolysis of amino acid ester and peptide synthesis from N-acyl amino acid ester and amino acid amide was investigated.

The K_m value of ester hydrolysis increased with an increase in the solvent content. Dioxane was the most effective and dimethyl sulfoxide (DMSO) the least, whilst K_cat showed a tendency to increase slightly in N, N-dimethylformamide (DMF) and DMSO. For dioxane and acetonitrile (MeCN) a maximum was observed.

In peptide formation from Fua-Phe-OEt and Gly-NH₂, dioxane and MeCN supported high product yield at molar fractions smaller than ca. 0.05 but the yield decreased significantly at higher fractions, although a relatively constant selectivity (ratio of the peptide bond formed to the ester consumed) was maintained. DMSO gave rather low peptide yields and selectivity even at lower molar fractions. DMF showed an intermediate tendency.

An apparent saturation parameter of the amine component was evaluated and the dissociation constant of a complex between acyl-enzyme and amino acid amide (K_n), as well as the rate constant of aminolysis exerted by the amino acid amide bound correctly on the enzyme (K_n), was calculated by initial rate analysis of peptide formation. In contrast to K_m values, K_n decreased with increasing concentrations of organic cosolvent. while a suppressive effect was observed (except for DMSO) on the K_n parameter.

Effects of the solvent practically immiscible in water was also studied by use of the enzyme physically “immobilized” on glass beads.     

Model-based characterization of an amino acid racemase from Pseudomonas putida DSM 3263 for application in medium-constrained continuous processes

The amino acid racemase with broad substrate specificity from Pseudomonas putida DSM 3263 was overproduced and characterized with respect to application in an integrated multi-step process (e.g., dynamic kinetic resolution) that--theoretically--would allow for 100% chemical yield and 100% enantiomeric excess. Overexpression of the racemase gene in Escherichia coli delivered cell free extract with easily sufficient activity (20-50 U mg(-1) total protein) for application in an enzyme membrane reactor (EMR) setting. Model-based experimental analysis of a set of enzyme assays clearly indicated that racemization of the model substrates D- or L-methionine could be accurately described by reversible Michaelis-Menten kinetics. The corresponding kinetic parameters were determined from progress curves for the entire suitable set of aqueous-organic mixtures (up to 60% methanol and 40% acetonitrile) that are eligible for an integrated process scheme. The resulting kinetic expression could be successfully applied to describe enzyme membrane reactor performance under a large variety of settings. Model-based calculations suggested that a methanol content of 10% and an acetonitrile content of 20% provide maximum productivity in EMR operations. However product concentrations were decreased in comparison to purely aqueous operation due to decreasing solubility of methionine with increasing organic solvent content. Finally, biocatalyst stability was investigated in different solvent compositions following a model-based approach. Buffer without organic content provided excellent stability at moderate temperatures (20-35 degrees C) while addition of 20% acetonitrile or methanol drastically reduced the half-life of the racemase.     

Peptide synthesis with immobilized carboxypeptidase Y

Enzymatic peptide synthesis was investigated using carboxypeptidase Y immobilized with glutaraldehyde on 10 mum microparticulate amino-silica. Carboxypeptidase Y was immobilized with 98.5% recovery of active enzyme to yield the immobilized enzyme having 0.55 units esterase activity/mg amino-silica support. The stability of the immobilized enzyme was examined as a function of pH, temperature, and reactant concentrations. Immobilized Carboxypeptidase Y was used in stirred batch and recirculating packed-bed reactors for peptide synthesis. Packed-bed reactors (40 x 4.6 mm, 60 x 4.6 mm) were used to catalyze the synthesis of 170 mg N-benzoyl-L-arginyl-L-methioninamide, 380 mg N-benzoyl-L-arginyl-L-methionyl-L-leucinamide, and 200 mg N-benzoyl-L-arginyl-L-methionyl-L-leucyl-L-phenylalaninamide in 8, 3, and 1 hour, respectively, as intermediates in the synthesis of L-methionyl-L-leucyl-L-phenylalanine. No inactivation of the immobilized enzyme was observed during the course of the reactions. The N-benzoyl-L-arginyl group served to increase the water solubility of the peptides and was removed by immobilized trypsin at the end of synthesis to obtain the final product. While the first two syntheses were conducted with aqueous reaction mixtures, the synthesis of N-benzoyl-L-arginyl-L-methionyl-L-leucyl-L-phenylalaninamide was carried out in a reaction mixture containing dimethylformamide to avoid precipitation of the product. HPLC and amino acid analysis confirmed the high purity and amino acid composition of the final product.     

A new approach to preparative enzymatic synthesis. Reprinted from Biotechnology and Bioengineering, Vol. XIX, No. 9, Pages 1351-1361

A new approach to preparative organic synthesis in aqueous-organic systems is suggested. It is based on the idea that the enzymatic process is carried out in a biphasic system "water-water-immiscible organic solvent." Thereby the enzyme is localized in the aqueous phase-this eliminates the traditional problem of stabilizing the enzymes against inactivation by a nonaqueous solvent. Hence, in contrast to the commonly used combinations "water-water-miscible organic solvent," in the suggested system the content of water may be infinitely low. This allows one to dramatically shift the equilibrium of the reactions forming water as a reaction product (synthesis of esters and amides, polymerization of amino acids, sugars and nucleotides, dehydration reactions, etc.) toward the products. The fact that the system consists of two phases provides another very important sources for an equilibrium shift, i.e., free energies of the transfer of a reagent from one phase to the other. Equations are derived describing the dependence of the equilibrium constant in a biphasic system on the ratio of the volumes of the aqueous and nonaqueous phases and the partition coefficients of the reagents between the phases. The approach has been experimentally verified with the synthesis of N-acetyl-L-tryptophan ethyl ester from the respective alcohol and acid. Porous glass was impregnated with aqueous buffer solution of chymotrypsin and suspended in chloroform containing N-acetyl-L-tryptophan and ethanol. In water (no organic phase) the yield of the ester is about 0.01%, whereas in this biphasic system it is practically 100%. The idea is applicable to a great number of preparative enzymatic reactions.     

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.     

Peptide synthesis catalyzed by polyethylene glycol-modified chymotrypsin in organic solvents

Chymotrypsin modified with polyethylene glycol was successfully used for peptide synthesis in organic solvents. The benzene-soluble modified enzyme readily catalyzed both aminolysis of N-benzoyl-L-tyrosine p-nitroanilide and synthesis of N-benzoyl-L-tyrosine butylamide in the presence of trace amounts of water. A quantitative reaction was obtained when either hydrophobic or bulky amides of L- as well as D-amino acids were used as acceptor nucleophiles, while almost no reaction occurred with free amino acids or ester derivatives. The acceptor nucleophile specificity of modified chymotrypsin as a catalyst in the formation of both amide and peptide bonds in organic solvents was quite comparable to that in aqueous solution as well as to that of the leaving group in hydrolysis reactions. By contrast, the substrate specificity of modified chymotrypsin in organic solvents was different from that in water since arginine and lysine esters were found to be as effective as aromatic amino acids to form the acyl-enzyme with subsequent synthesis of a peptide bond.     

Enzymatic reactions in aqueous-organic media. VI. Peptide synthesis by α-chymotrypsin in hydrophilic organic solvents

The peptide synthesis from N-acetyl-L-tyrosine ethyl ester and amino acid amides was realized using α-chymotrypsin as a catalyst in ethanol or acetonitrile containing small amounts of water. In these reaction systems, the precipitates of phosphate salt, which was used as a component of buffer solution, are considered to act as carriers of chymotrypsin. It was found that peptide formation is competitive with hydrolysis of the substrate ester, but the secondary synthesis of the peptide from the hydrolysate was also considered to proceed. The yield of the peptide after 24 h reaction was strongly dependent on the water concentration; maximum yields of the peptide were obtained at water concentrations below 10% (v/v). The addition of tertiary amines, such as triethyl amine, markedly increased the peptide yield, probably due to the increase in the concentration of the nucleophilic amine components by neutralization of hydrohalides of amino acid amides. The effect of reaction temperature and the reactions with CT immobilized on PVA, chitosan, or TEAE-cellulose are also described.     

Bacillus licheniformis protease-catalyzed peptide synthesis via the kinetically controlled approach using the carbamoylmethyl ester as an acyl donor in anhydrous acetonitrile

Summary The couplings ofN-protected amino acid esters with amino acid amides proved to be carried out in anhydrous acetonitrile in the presence ofBacillus licheniformis protease (subtilisin Carlsberg) immobilized on Celite. The maximal peptide yields were obtained with the immobilized enzyme prepared through lyophilization from a pH 10.7 buffer solution. A series of dipeptide syntheses and several segment condensations were achieved generally in high yields by the combined use of the immobilized enzyme prepared from this pH and the carbamoylmethyl ester as the acyl donor.     

Bacillus licheniformis protease-catalyzed peptide synthesis via the kinetically controlled approach using the carbamoylmethyl ester as an acyl donor in anhydrous acetonitrile

The couplings of N-protected amino acid esters with amino acid amides proved to be carried out in anhydrous acetonitrile in the presence of Bacillus licheniformis protease (subtilisin Carlsberg) immobilized on Celite. The maximal peptide yields were obtained with the immobilized enzyme prepared through lyophilization from a pH 10.7 buffer solution. A series of dipeptide syntheses and several segment condensations were achieved generally in high yields by the combined use of the immobilized enzyme prepared from this pH and the carbamoylmethyl ester as the acyl donor.     

Stabilization of substilisin E in organic solvents by site-directed mutagenesis

Subtilisin E was rationally engineered to improve its stability in polar organic solvents such as dimethylformamide (DMF). A charged surface residue, Asp248, was substituted by three amino acids of increasing hydrophobicity, Asn, Ala, and Leu; all three variants were stabilized with respect to wild type in 80% DMF. This stabilization was only observed in the presence of high concentrations of the organic solvent: no stability enhancements were observed in 40% DMF. In contrast, the mutation Asn218 --> Ser alters internal hydrogen bonding interactions and stabilizes subtilisin E in both 40% and 80% DMF. This study provides additional evidence that substitution of surface-charged residues is a generally useful mechanism for stabilizing enzymes in organic media and that the stabilizing effects of such substitutions are unique to highly altered solvent environments. The effects of the single amino acid substitutions on free energies of stabilization are additive in the Asp248 --> Asn + Asn218 --> Ser combination variant, yielding an enzyme that is 3.4 times more stable than wild type in 80% DMF.     

Surfactant-protease complex as a novel biocatalyst for peptide synthesis in hydrophilic organic solvents*

The peptide synthesis from N-acetyl-L-phenylalanine ethyl ester with alaninamide catalyzed by a surfactant-protease complex has been performed in anhydrous hydrophilic organic solvents. Proteases derived from various sources were converted to surfactant-coated complexes with a nonionic surfactant. The surfactant-subtilisin Carlsberg (STC) complex had a higher enzymatic activity than the other protease complexes and the initial reaction rate in tert-amyl alcohol was 26-fold that of STC lyophilized from an optimum aqueous buffer solution. Native STC hardly catalyzed the same reaction. The addition of water to the reaction medium activated the lyophilized STC, however, the reaction rate was much lower than that of the STC complex, and a hydrolysis reaction preferentially proceeded. The STC complex exhibited a high catalytic activity in hydrophilic organic solvents (e.g. tertiary alcohol). The addition of dimethylformamide as a cosolvent improved the solubility of amino acid amides and further activated the STC complex due to the water mimicking effect. When hydrophilic amino acid amides were employed as an acyl acceptor, the peptide formation proceeded efficiently compared to that using hydrophobic substrates. The surfactant-STC complex is a powerful biocatalyst for peptide synthesis because the STC complexes display a high catalytic activity in anhydrous hydrophilic organic solvents and did not require the excess amount of water. Thus the side (hydrolysis) reaction is effectively suppressed and the yield in the dipeptide formation is considerably high.     

Catalytic activity and stability of xanthine oxidase in aqueous-organic mixtures

In the present study, bovine milk xanthine oxidase activity in various aqueous-organic mixtures and the effects of pH, temperature, and lyophilization on the enzyme activity have been investigated. The enzyme was incubated with xan-thine as the substrate in Sorenson’s phosphate buffer (pH 7.0) containing 0.1 mM EDTA, and the activity was determined spectrophotometrically in the absence and presence of different fractions of nine water-miscible organic solvents at 27–50°C and at different pH values ranging from 6 to 9. The organic solvents reduced the enzyme activity to different extents. In spite of these inhibitory effects, the enzyme showed relatively good stability in the aqueous-organic mixtures compared with the aqueous medium. A significant increase in the activity of the lyophilized enzyme was observed in pure organic solvents. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 1, pp. 124–130.     

Peptide synthesis in organic media with subtilisin 72 immobilized on poly(vinyl alcohol)-cryogel carrier

Serine proteinase subtilisin 72 was covalently attached to the beads of poly(vinyl alcohol)-cryogel, a macroporous hydrogel prepared by the freeze-thaw technique. The immobilized enzyme was examined as a catalyst in the synthesis of protected peptides Z-Ala-Ala-Xaa-Phe-pNA (Xaa = Leu, Glu, Lys) in acetonitrile/dimethylformamide mixtures. Immobilized subtilisin catalyzed with high yield the formation of peptide bonds between Phe-pNA and acyl donors including those with free carboxylic group and non-protected C-terminal basic and acidic amino acid residues.