Chymotrypsins Modified by Neutral or Charged Amphiphiles as Catalysts for Ester Synthesis in Polar Organic Solvents

-Chymotrypsin has been modified by a series of neutral liposaccharidic or charged lipocarboxylic amphiphile reagents. In the esterification of N-acetyl tyrosine in three polar solvents, the new biocatalysts have been compared to chymotrypsins modified by reductive alkylation with glyoxylic acid, melibiose or octanal. This comparison indicates that the rate accelerations observed with the neutral or anionic amphiphile-coated enzymes are mainly due to the hydrophobization of the protein surface in the neigbourhood of the external lysine residues. This interpretation is strengthened by the favorable effect of supports more hydrophobic than celite on the reaction.     

Peptide Synthesis Catalyzed by Crosslinked α-Chymotrypsin in Water/Dimethylformamide Solvent System

-Chymotrypsin was crosslinked to give a water-insoluble polymer by treatment with the bifunctional reagent glutaraldehyde. The specific activity of the crosslinked enzyme in aqueous media was three orders of magnitude lower than for the native chymotrypsin. In a medium containing more than 50% (v/v) of dimethylformamide the specific activities of both enzymes were comparable. In addition, the insoluble polymer was more stable in the presence of 60% (v/v) dimethylformamide compared with the native enzyme. Therefore, in this medium enzymatic peptide synthesis could be successfully accomplished with the crosslinked enzyme, but not with the same amount of native chymotrypsin.     

Effects of subzero temperatures on the kinetics of protease catalyzed dipeptide synthesis in organic media

A depeptide synthesis was drastically influenced by the reaction temperature, in the range from -30 degrees to 25 degrees C. This article shows the kinetic reasons of this effect. alpha-Chymotrypsin was immobilized on celite and used in four different water-miscible solvents containing small amounts of water-miscible solvents containing small amounts of water. The reaction studied was the aminolysis of N-acetyl-L-phenylalanine ethyl ester (Ac-PheOEt) with L-alaninamide (Ala-NH(2)) and water for the acylenzyme complex, the nucleophile was favoured by low reaction temperatures. This effect (quantified as p-values) was observed in all four solvents, and it was greatest in acetonitrile and tetrahydrofuran. The esterase and amidase activities of the enzyme were studies using AcPheOEt and N-acetyl-L-phenylalanyl-L-ananinamide (AcPheAla-NH(2)) as substrates. The Michaelis-Menten parameters, K(m,app) and V(max), were determined for ester hydrolysis and dipeptide hydrolysis. Both K(m,app) and V(max) tended to increase with increasing temperature. Secondary hydrolysis was reduced at subzero temperatures because ester hydrolysis was favoured in relation to depeptide hydrolysis. Depeptide synthesis was thus favored by low temperatures in two ways: first, in the competition between the nucleophile and water for the acyl enzyme; and, second, in the competition between the ester substrate and the peptide substrate for the free enzyme. As a result, in acetonitrile containing 10% water, the maximal yield was 99% at -20%C compared with 84% at 25 degrees C. (c) 1995 John Wiley & Sons, Inc.     

Reactions Catalyzed by Peg-Modified α-Chymotrypsin in Organic Solvents. Influence of Water Content and Degree of Modification

-Chymotrypsin was modified with cyanuric chloride activated monomethoxypolyethylene glycol (MPEG) with molecular weights 1900 and 5000. Using the higher molecular weight MPEG a product that was soluble in benzene at moderate levels of modification was obtained, whereas with MPEG 1900 almost all the enzyme's amino groups had to be modified for dissolving the conjugate. The catalytic activity decreased with increasing degree of substitution. Apparent V_max was considerably higher for the less modified enzyme preparation than for the more modified one, while K_m,app stayed almost constant. The modified enzyme was used for peptide synthesis. The reaction was dependent on the content of dissolved water. Both V_max,app and K_m,app increased with increasing water content. It was possible to achieve a process with complete conversion of substrate to dipeptide.     

α-Chymotrypsin Immobilized on Chitin. Relationships Between the Enzyme Kinetic Constant and Support Structure

-Chymotrypsin was immobilized on chitin from squills, lobsters and prawns by means of glutaraldehyde. Hydrolase and peptide synthetase activities were determined in aqueous and homogeneous aqueous-organic media, respectively.

The results show -chymotrypsin immobilized on chitin from prawn to be the most active immobilized derivative based on its synthetase activity (90% yield of Bz-Tyr-Leu-NH₂ in carbonate buffer, p^H 9 containing 70% 1,4- butanediol).

The relationship between the kinetic constant of hydrolysis and chitin structure was also studied. -Chymotrypsin immobilized on prawn chitin was found to be the best derivative in kinetic terms.

The stability of the three derivatives was studied at 37C.     

Synthesis of Oligosaccharides by β-Fructofuranosidase in Biphasic Medium Containing Organic Solvent as Bulk Phase

The effect of four organic solvents on β-fructofuranosidase mediated synthesis of oligosaccharides from sucrose were investigated. Amongst the solvents examined, butyl acetate proved to be the best for oligosaccharide synthesis. Starting with the equivalent of 44.6 g/L of sucrose, 247 U of enzyme and 91.6% (by vol.) of butyl acetate results in the production of 8.8 g/L of oligosaccharides within 30 min, with trisaccharides constituting more than 60% of the oligosaccharides. The efficiency for conversion of sucrose to oligosaccharides is greater than 19%, and this exceeds the 11.6% (in 24 h) previously achieved with 1271 U of the same enzyme in aqueous medium. Use of butyl acetate as the bulk phase therefore modifies the reaction environment in favour of enhanced and accelerated rate of oligosaccharide synthesis by this β-fructofuranosidase.     

Transesterification catalyzed by polystyrene-supported chymotrypsin in toluene: the effect of neutralization of basic or acidic groups attaching to polystyrene resins

Crosslinked polystyrene resins containing a low level of either basic or acidic groups were used for supports of alpha-chymotrypsin (CT), which catalyzed the transesterification of N-acetyl-L-phenylalanine ethyl ester (AcPheOEt) with propanol in toluene. With a minimal amount of water, CT was sorbed to the resins, basic or acidic groups of which were partly or fully neutralized by several soluble acids or bases. With an increasing degree of neutralization of basic resins by free acids, the rate of disappearance of AcPheOEt was decreased, whereas the by-product formation of AcPheOH, due to hydrolysis, was considerably suppressed, compared with the ester-exchange product, AcPheOPr. The pK(a) value of the neutralizing acid was also important for both CT activity and reaction selectivity. AcPheOPr was selectively produced at a certain range of pK(a) values. On the other hand, the neutralization of acidic resins with free amines enhanced the CT activity but a strong base promoted the formation of hydrolysis product. (c) 1995 John Wiley & Sons, Inc.     

Salt-Enhanced Aminolysis of Acyl-α-Chymotrypsins by Dipeptides

Increase in KC1 concentration from 0.1 m to 3 m enhances the chymotrypsin-catalyzed acyl-transfer to a series of dipeptides, H₂N—CH(R₁)C(O)NHCH(R₂)C(O)O, by factors between 15 and 44. The observed positive salt effect seems to be a screening of the unfavorable electrostatic interaction between the dipeptide carboxyl group and a negatively charged area in the S' subsite on the enzyme surface. The effect is of practical use in preparative peptide synthesis—in 3 m KCl solutions the analytical yields of tripeptides of up to 99 per cent have been obtained.     

Refolding of a denatured α-chymotrypsin and its smart bioconjugate by three-phase partitioning

-Chymotrypsin inactivated with 8 M urea and 100 mM dithiothreitol could be completely reactivated by subjecting it to three-phase partitioning (TPP). TPP consisted of adding 30% w/v ammonium sulfate and t-butanol (volume equivalent to aqueous solution of denatured -chymotrypsin) at 25°C. The activated -chymotrypsin was recovered as an interfacial precipitate between the upper organic and lower aqueous phase. It was found that this could be extended to a thermally inactivated smart bioconjugate of -chymotrypsin with Eudragit S-100 (a reversibly soluble-insoluble methmethacrylate). The thermally inactivated bioconjugate had to be further subjected to urea and dithiothreitol before refolding by three-phase partitioning. Ninety per cent of the activity of the bioconjugate could be recovered. The free enzyme and its bioconjugate which lost activity in the presence of 90% dioxane recovered 94 and 90% of their activities, respectively, by employing TPP. The refolded free enzyme and its bioconjugate were evaluated in terms of V _max/K _m and their fluorescence emission spectra.     

Protein extraction by reverse micelles: a study of the factors affecting the forward and backward transfer of alpha-chymotrypsin and its activity

alpha-chymotrypsin is taken as a model protein to investigate three aspects of the protein extraction by reverse micelles: (1) the comparison between the two forward transfer techniques, i.e., the liquid-liquid and the solid state-liquid transfer; (2)the back-transfer, i.e., the capability of the protein to be recovered from the micellar solution; and (3) the maintainance of the enzyme activity at the end of the extraction cycle. Concerning the forward transfer from the liquid phase, we study first the effect of salt initially present in the aqueous phase on the equilibrium concentration of the extracted species; further, we study the forward protein extraction from the solid state, and the effect of pH, salt, and protein concentration on the transfer efficiency. Concerning the back transfer, we find the somewhat surprising result, that the percentage of protein back-extraction depends on the type and concentration of salt used for the forward transfer. Preliminary data concerning an alternative method for the back-transfer using silica gel to liberate the protein from the micellar environment, are presented. Finally, it is found that the enzyme activity depends again on the type and concentration of salt used for the forward transfer.     

Controllable Regioselective Acylation of Rutin Catalyzed by Enzymes in Non-aqueous Solvents

An efficient route to synthesize 3′′- and 4′′′-vinyl rutin esters has been developed by enzyme-catalyzed regioselective acylation of rutin with divinyl dicarboxylates in organic media. Alkaline protease from Bacillus subtilis provided 3′′-O-substituted vinyl rutin esters in pyridine, and Novozym 435 gave 4′′′-O-substituted vinyl rutin esters in tert-butanol.     

α-Chymotrypsin shows higher activity in water as well as organic solvents after three phase partitioning

Alpha-Chymotrypsin was found to show a 119% increase in activity after three phase partitioning. The k_cat/K_m of the partitioned enzyme (TPP-C) for hydrolysis of Bz-Tyr-OEt in aqueous medium at 25°C was found to be 48.3×10⁴ mM^-1 min^-1 as compared to the corresponding value of 17.7×10⁴ mM^-1 min^-1 for the untreated control (C). The λ_max of the fluorescence emission spectrum of TPP-C showed 178% increase in the quantum yield when compared to C. TPP-C showed a 2.94 and 3.58 fold increase (as compared to C) in initial rates for formation of the ester Ac-Phe-OEt (from Ac-Phe and ethanol) in low water containing toluene and n-octane, respectively. It was found that TPP-C also showed the phenomenon of pH memory. At 5% (v v^-1) water (in t-amyl alcohol), while no esterification was observed with C, TPP-C still showed significant level of esterification activity.     

Enzymatic Synthesis of Polyol- and Masked Polyol- Glycosides using β-Glycosidase of Sulfolobus Solfataricus

The use of commercially available mesophilic glycosidases in the enzymatic synthesis of glycosides of different types is a well established method suffering from some drawbacks such as a poor yield. Substrates with three or four hydroxyl groups have been subjected to enzymatic glucosylation using crude homogenate of the thermophilic archaeon Sulfolobus solfataricus containing a β-glycosidase activity able to transfer glucose, galactose and fucose from different donors. The stereochemistry of this reaction was interpreted in terms of interaction with a possible “glucose” active site of the enzyme. In addition masked or protected derivatives of tetritols and some simple unsaturated alcohols were glycosylated yielding glycosides in yields very competitive with those obtained using mesophilic enzymes, examples of further chemical manipulation of these compounds were reported. When using a scarce amount of acceptor, a reasonable amount of products could be obtained by adding different aliquots of donor at time intervals.     

Comparison of the Synthetic and Hydrolytic Activity of Immobilised Cephalexes-Synthetase

The synthesis of semisynthetic β-lactam antibiotics from 7-amino-cephalosporanic or 6-amino-penicillanic acids and phenyl-glycine esters is catalysed by immobilised cephalexin-synthetase The synthetic activity of the biocatalyst correlates with its activity in the hydrolysis of the phenyl-glycine esters.     

The aminoterminal domain of alpha-tubulin probed by monoclonal antibodies. Recognition of a rarely exposed epitope by the monoclonal antibody 111 B52 C2

The production and identification of a monoclonal antibody, 111 B52 C2, raised against fragments obtained after limited proteolysis of purified tubulin is described. The recognized epitope is located on the aminoterminal domain of the alpha-tubulin subunit and differs from the antigenic sites reacting with the presently existing panel of available monoclonal antibodies. This monoclonal antibody thus constitutes a potentially useful tool to explore interactions between tubulin and other specific ligands.     

Synthesis of AcPheLeuNH(2) by alpha-chymotrypsin in TTAB reversed micelles: Application of response surface methodology to the optimization of the system

The influence of the long chain alcohols, hexanol, octanol, and decanol, as cosurfactants of the reverse micellar system of tetradecyltrimethylammonium bromide on the alpha-chymotrypsin-mediated AcPheLeuNH(2) synthesis was studied. The effect of temperature, buffer molarity, pH, and substrate concentration was also evaluated. The enzyme was chemically modified and the effect of this modification upon the enzyme activity was also analyzed. Octanol allowed a higher activity for both enzyme forms. The peptide synthesis/substrate hydrolysis ratio is independent of the long chain alcohol used. The chemical modification decreases the alpha-chymotrypsin activity under the system conditions studied, but increases the initial velocity of peptide synthesis relative to the ester substrate hydrolysis. The response surface methodology was applied to optimize the dipeptide synthesis in the system containing octanol as cosurfactant. (c) 1994 John Wiley & Sons, Inc.     

An Insight into the Active Site of Pseudomonas Fluorecens (P. cepacia) Lipase to Define the Stereochemical Demand for the Transesterification in Organic Solvents

The Pseudomonas fluorescens lipase-catalyzed transesterification of 2-methyl alkanols 1 and the 2-substituted oxiranemethanols 2 with vinyl acetate in organic solvents has been studied and the results discussed in terms of steric and electronic demand within the recently postulated models of the lipase active site.     

Enzymatic Peptide Synthesis in Frozen Aqueous Solution: Use of Nα-unprotected Peptide Esters as Acyl Donors

The ability of the endopeptidase α-chymotrypsin (EC 3.4.21.1) to catalyse the reaction of various N^α- unprotected di- and tripeptide ester derivatives with H-Leu-NH₂, and with a series of C-terminal free di- and tripeptides at −15° C in frozen aqueous solution was investigated. The enzyme is able to synthesize N- and C-terminal unprotected penta- and hexapeptides in up to 92% yield, depending on the amino component used, in a single-step segment-condensation reaction. Freezing the reaction mixture resulted in significantly increased peptide yields compared with the reaction at room temperature. The enzyme shows a modified nucleophilic specificity in frozen solution compared with room temperature. Nucleophilic amino components with positively charged amino acids in P₂^′ -position are accepted. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Enzymatic Peptide Synthesis in Organic Media. Synthesis of CCK-8 Dipeptide Fragments

The enzymatic synthesis of the seven consecutive dipeptide fragments of the cholecysto kinin C-terminal octapeptide (CCK-8) in organic media is reported. The influence of the reaction medium composition, the protease, and the structure of N-α and C-α protecting groups of both carboxyl and amino components was evaluated. α-Chymotrypsin, papain and thermolysin adsorbed on Celite were used as catalysts, under thermodynamic and kinetic control. The carboxamidomethyl, methyl and allyl ester derivatives of acetyl, benzyloxycarbonyl, tert-butyloxycarbonyl and fluoren-9-ylmethoxycarbonyl amino acids, were assayed as carboxy components. Amino acid amide and ester derivatives were employed as nucleophiles with a preference for the latter, since the dipeptide product obtained could be used directly, without any further chemical modification, as acyl-donor in subsequent coupling steps. All dipeptides selected were successfully synthesized, using the optimal combination of protecting groups, reaction media and enzyme different for each coupling reaction. The information gained with this study should be instrumental in designing an optimal strategy for the total enzymatic synthesis of cholecystokinin C-terminal octapeptide (CCK-8).     

Enzymatic Routes for the Synthesis of Rhododendrin and Epi-Rhododendrin

The synthesis of (R)- and (S)-3-(4-hydroxyphenyO-1-methylpropyl-β-D-glucopyranosides has been achieved by two enzymatic steps, namely an oxido-reduction step involving alcohol dehydrogenases from different origin for the preparation of both aglycones in enantiomeric pure form, and a transglycosidation step involving a thermophilic β-glucosidase from the archaeon Sulfolobus solfataricus.