Chymotrypsin aqueous solution

For many solubilized enzymes the greatest catalytic activity and/or changes in conformation are found at R < 12, namely, when the competition for the water in the system between surfactant head groups and biopolymers is strong. This emphasizes the importance of the hydration water surrounding the biopolymer on its reactivity and conformation [13], It has been reported that enzymes incorporated in the aqueous polar core of the reversed micelles are protected against denaturation and that the distribution of some proteins, such as chymotrypsine, ribonuclease, and cytochrome c, is well described by a Poisson distribution. The protein state and reactivity were found markedly different from those observed in bulk aqueous solution [178,179],... 

For the purpose of synthesizing flavor peptides or proteins in large scale, we developed "protein recombination method" and "enzymatic synthesis using chemically modified enzyme". "Protein recombination method" was applied to the synthesis of C-terminal portion of p-casein and its analog. Chymotrypsin was chemically modified by Z-DSP in aqueous solution. It was stable for organic solvents. Using this modified enzyme, we succeeded in the synfiiesis of Inverted-Aspartame-Type Sweetener "Ac-Phe-Lys-OH" in one step. 

The initial evidence for the formation of an acyl-enzyme ester intermediate came from studies of the kinetics with which chymotrypsin hydrolyzed analogs of its normal polypeptide substrates. The enzyme turned out to hydrolyze esters as well as peptides and simpler amides. Of particular interest was the reaction with the ester p-nitrophenyl acetate. This substrate is well suited for kinetic studies because one of the products of its hydrolysis, p-nitrophenol, has a yellow color in aqueous solution, whereas p-nitrophenyl acetate itself is colorless. The change in the absorption spectrum makes it easy to follow the progress of the reaction. When rapid-mixing techniques are used to add the substrate to the enzyme, an initial burst of p-nitrophenol is detected within the first few seconds, before the reaction settles down to a constant rate (fig. 8.8). The amount of p-nitrophe-... 

Catalytic activities of a-chymotrypsin and Subtilisin Carlsberg in various hydrous organic solvents were measured as a function of how the enzyme suspension had been prepared (Ke, 1998). Direct suspension of the lyophilized enzyme in the solvent containing 1% water was compared with precipitation of the same enzyme from its aqueous solution by a 100-fold dilution with anhydrous solvent. The reaction rate in a given non-aqueous enzymatic system was found to depend on the nature of both enzyme and solvent, but to depend strongly on the mode of enzyme preparation. 

Usually oxime has a high nucleophilicity in dissociated form, but it is useless in a moderate aqueous solution for its high pK value (pK = 11 12). The oxime moiety which is bound to poly(4-vinylpyridine) by the quartemization shows a lower pK value (pK = 8.5 0.3) and high nucleophilicity in the hydrolysis of PNPA(5) (95). The catalytic activity of the polymer(X) reaches a half of that of a-chymotrypsin showing ka = 200 M-1 sec-1 at pH = 8, where m is 12. 

I Cyclodextrins are excellent enzyme models Catalysis and induced fit. Due to their cavities, which are able to accommodate guest (substrate) molecules, and due to the many hydroxyl groups lining this cavity, cyclodextrins can act catalytically in a variety of chemical reactions and they therefore serve as good model enzymes. Thus, benzoic acid esters are hydrolyzed in I aqueous solution by factors up to 100 times faster if cyclodextrins are added. The reaction in- j volves an acylated cyclodextrin as intermediate which is hydrolyzed in a second step of the j reaction, a mechanism reminiscent of the enzyme chymotrypsin. The catalytic efficiency can. be further enhanced if the cyclodextrins are suitably modified chemically so that a whole range of artificial enzymes have been synthesized [551-555, 556, 563, 564]. 

The search for an enzymatic activity that would catalyze prolyl peptide bond isomerization began soon after the proposal of the proline hypothesis. The success came in 1984, when Fischer and co-workers discovered a peptidylprolyl m—tram-isomerase activity in porcine kidney and other tissues by an assay that is based on the conformational specificity of chymotrypsin. This protease cleaves the 4-nitroanilide moiety from the peptide glutaryl-Ala-Ala-Pro-Phe-4-nitroanilide only when the Ala-Pro peptide bond is in the trans conformation. In aqueous solution 90% of the molecules are trans in the assay peptide and only 10% are cis. Therefore, in the presence of a high concentration of chymotrypsin, 90% of the hydrolysis reaction occurs within the dead time of manual mixing. Hydrolysis of the remaining 10% is slow, limited in rate by the cis — ... 

The properties of a protein in a reverse micelle depend strongly on water content. Typically, at mole ratios of water to surfactant (wo) of less than about 3, there is no enzyme activity. As Wg is increased the activity sharply rises, sometimes to an optimum value at Wg — 5-20. The value of kcat for chymotrypsin is as much as 5-fold greater in AOT reverse micelles than it is in aqueous solution (Barbaric and Luisi, 1981 Fletcher... 

Figure 1 Comparison of infrared spectra of a-chymotrypsin in aqueous solution and dried solid state. The insert shows the second derivatives in the amide I region for the spectra in the main panel. (From Ref. 11.)...

Stock solutions of DFP can be conveniently prepared in isopropanol in concentrations from 0.1 M to 0.001 M. These solutions are stable for a month in the refrigerator (Jansen et al. 1949). Moon et al. (1965), in their studies of the reaction of chymotrypsin with DFP, have determined the normality of stock solution of DFP in the following manner. Roughly 0.03 moles of DFP were added to an aqueous solution of 0.17 moles of KOH in a volumetric flask. The solution was allowed to stand for 12 hr at 25°C to permit complete hydrolysis, and was titrated to pH 7.0 with 0.1 N HCl. Identical titrimetric results were obtained after 43 hr standing in alkali indicating that complete hydrolysis had taken place after 12 hr. To determine the amount of free acid, if any, in the DFP, the same amount of DFP that was used... 

The rate of a-chymotrypsin-catalyzed hydrolysis as a function of overall GPANA concentration in CTAB reversed micelles and in aqueous solution are shown in Figure 5. It is apparent that the reaction rate in the reversed micellar solution is on the order of 50 times more rapid than in the aqueous system. Furthermore, in the reversed micellar system there is no indication of enzyme saturation as the reaction is first order in substrate concentration. As enzyme saturation kinetics are not observed, it is impossible to differentiate between the parameters kcat and Kg. Instead a second order bimolecular rate constant for both the micelle interior ( micelle) and for what is experimentally observed ( observed) is defined. 

Salam, S.M.A., Kagawa, K., and Kawashiro, K. (2006) alpha-Chymotrypsin-catalyzed peptide synthesis in frozen aqueous solution using N-protected amino acid carbamoylmethyl esters as acyl donors. Tetrahedron Asymmetry, 17 (1), 22-29. 

Kinetic analysis has even been attempted for the degradation of peptide and protein pharmaceuticals for which the mechanism and pathways are unknown. Apparent inactivation of a-chymotrypsin and bromelain in aqueous solutions was described by monoexponential... 

Figure 208. First-order plots of inactivation of a-chymotrypsin (a) and bromelain (b) in aqueous solution at various temperatures (pH 7.4). (Reproduced from Ref. 879 with permission.)...

FIG. 6 Dependence of the first-order rate constant (keat) for deacylation of N-trans-cinnamoy -a-chymotrypsin ( ), as well as rotational frequency, v (O), and hyperfine splitting constant, a (A) for spin-labeled a-chymotrypsin on degree of hydration (water-to-AOT molar ratio) in the system AOT (0.1 M)-water-octane. Spin label 2,2,5,5-tetramethyl-4-iodoacetamidopyrrolidine-l-oxyl. Dashed lines show keat and a values in aqueous solution. (From Ref. 41.)... 

FIG. 7 Dependence on the water/surfactant molar ratio of (a) the maximal reaction rate normalized to the enzyme concentration, V/E, of a-chymotrypsin-catalyzed hydrolysis of N-benzoyl-L-tyrosine p-nitroanilide, and (b) the rotational frequency, v, of the spin label in the active site of the enzyme in the system AOT-water/glycerol-octane. Water/glycerol volume ratios 1—100 0 2—80 20, 3—50 50, 4—20 80, 5—6 94. Dashed lines show V/Eq and v values in aqueous solution. (From Ref. 42.)... 

FIG. 11 Combination of two factors regulating enzyme catalytic activity variation in the surfactant concentration and addition of water-miscible organic solvents, (a) Peroxidase in the system AOT-water/glycerol-octane at water/glycerol volume ratios (O) 100 0 ( ) 20 80. (b) a-Chymotrypsin in the system AOT-water/glycerol-octane at water/glycerol volume ratios (O) 100 0 ( ) 6 94. Dashed lines show the catal5dic activities of the enzymes in aqueous solution. (From Ref. 44.)... 

Furthermore, the stability of a-chymotrypsin in [EMIM][(CF3S02)2N] was studied by De Diego et al. Results were compared to those obtained in 1-propanol, a deactivating medium, and an aqueous solution of sorbitol, an enzyme-stabihzing medium. Using fluorescence and circular dichroism studies they showed that of the solvents used only the ionic liquid was able to stabilize the enzyme via the formation of a flexible and more compact 3D structure [41]. 

Next, in Section 8.3.2, we describe long-time full atomic MD simulations (longer than 150 ns) for a small peptide, met-enkephalin (M-Enk), in addition to a larger chymotrypsin inhibitor 2 (CI2), both in ectoine aqueous solutions with the same concentration and in pure water at room temperature. To determine the spatial distribution of each solvent component, the atom number densities of water molecules and ectoine molecules around both solutes were analyzed. We found that one dominant structure of M-Enk does not preferentially exclude the ectoine molecules from its surface as CI2 does. To understand the reason for this difference in ectoine exclusion, in addition to the effect of direct interaction between M-Enk and ectoine, the influence of hydration (i.e., property alteration of the hydration layer near the solute surface) on the development of ectoine preferential exclusion around each solute was examined at the molecular level. 

Chymotrypsin Inhibitor 2 and Met-Enkephalin in Ectoine Aqueous Solution... 

The pH dependence of enzymatic activity in microemulsions sometimes differs from that in aqueous solution. For instance, several groups found that for a-chymotrypsin-catalyzed hydrolysis the optimum pH is shifted to considerably more alkaline conditions in the... 

Silicone polymers, among the most hydrophobic species known [1], are often deleterious to protein structure. For example, shaking an aqueous solution of a-chymotrypsin with D4 (octamethylcyclotetrasiloxane) for a few minutes leads to about 85 % loss of enzymatic activity [2]. It was surprising, therefore, to learn that the presence of only a few hydrophilic, functional groups on a silicone surfactant can dramatically both stabilize the emulsion and, in some cases, decrease the rate of denaturation of the enzyme, as measured by changes in enzyme activity [3]. 

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