Catalytic Activity Of Α-chymotrypsin Research Articles

Enhanced Catalytic Activity of α-Chymotrypsin in Cationic Surfactant Solutions: The Component Specificity Revisited.

Enhanced catalytic activity (super activity) of enzymes in the presence of surfactants is of key importance in "micellar enzymology"; such super activity is not very trivial, it is highly system specific, and the mechanism behind the activity enhancement is not always well apprehended. We report the catalytic activity of α-chymotrypsin (CHT) on ala-ala-phe-7-amido-4-methylcoumarin (AMC) in the presence of cationic surfactants of different hydrophobic chain lengths: dodecyltrimethylammonium bromide (DTAB), cetyltrimethylammonium bromide (CTAB) and octadecyltrimethylammonium bromide (OTAB). It is observed that in comparison to buffer the catalytic activity of CHT is enhanced 5-fold in premicellar DTAB solutions, while negligible changes are observed in CTAB and OTAB. Activity decreases considerably in the post micellar concentration, specifically for the latter two surfactants. A similar trend is also obtained in another substrate 2-napthyal acetate hydrolysis. Such surfactant specific superactivity is intriguing. The protein's secondary and tertiary structures in the presence of these surfactants are determined using circular dichroism (CD) spectroscopy and it is found that both CTAB and OTAB perturb the protein structure significantly, especially in the post micellar concentrations. DTAB, on the other hand, does not produce noticeable changes in the protein structure. The various pairwise interactions present in the system have been underlined using both steady-state and time-resolved fluorescence spectroscopy. Assuming a three-step kinetics model, we determine the free energy changes of the reaction, and the observations have been discussed in the light of the various interactions among the components.

Influence of Amine-Based Cationic Gemini Surfactants on Catalytic Activity of α-Chymotrypsin

The α-chymotrypsin activity was tested in aqueous media with the presence of novel cationic amine–based gemini surfactant, with different spacer chain lengths and head group size, and also compared with the cationic cetyltrimethylammonium bromide (CTAB) and cetyltriphenylphosphonium bromide (CTPB) surfactants and aqueous buffer only. The p-nitrophenyl acetate (PNPA) hydrolysis rate was monitored in the presence of the surfactant concentration at 30°C. Most of these gemini surfactants gave higher catalytic activity as compared to cationic CTAB and CTPB. The highest superactivity was measured in the presence of gemini 16-12-16, [dodecanediyl-1,12-bis(cetyldimethylammonium bromide)] surfactant at pH 7.5. The catalytic reaction follows the Michaelis–Menten mechanism. The catalytic rate constants, kcat, show the same profile that the catalytic affinity; KM being enhanced with increasing space chain length. The results are favorable for considering that the amine-based gemini surfactant influences more than both the aqueous and cationic micellar media.

Synthesis of 2,3-dihydroquinazolin-4(1H)-ones catalyzed by α-chymotrypsin

We discovered that α-chymotrypsin has a promiscuous ability to catalyze the cyclocondensation of aromatic and aliphatic aldehydes with 2-aminobenzamides to afford the corresponding 2,3-dihydroquinazolin-4(1H)-ones successfully in high yields (90%–98%) under alcohol solvent. The catalytic activity of α-chymotrypsin was evaluated through investigating the temperature, the enzyme loading and the ratio of substrates in the enzyme-catalyzed reactions. The present method proves to be efficient and environmentally friendly in terms of short reaction time, high yield, green catalyst and the clean products obtained without further purification processes.

Structure and catalytic activity of α-chymotrypsin in solutions of gemini surfactants

The regulatory effect of gemini alkylammonium surfactants (GSurf) with the hexamethylene spacer varying in the length of alkyl radicals on the structure and catalytic activity of a-chymotrypsin was studied. A correlation between the activity of a-chymotrypsin and the length of the alkyl radical of GSurf was found. Gemini surfactants enhance the enzyme activity below the critical micelle concentration (CMC) and inhibit that above the CMC. The results of IR spectroscopy and the data on tryptophan fluorescence show that the interaction of GSurf with a-chymotrypsin induces changes in the protein structure differed in intensity. The most probable enzyme complexes with GSurf were characterized by the molecular docking method.

Effect of chemical composition of SBA-15 on the adsorption and catalytic activity of α-chymotrypsin

The adsorption of α-chymotrypsin (α-CT) on Al-containing mesoporous materials based on SBA-15 topology was investigated. The insertion of various amounts of aluminium was achieved using the two-step (pH-adjusting) method and the physico-chemical properties of these materials were characterized by small angle X-ray diffraction, nitrogen adsorption/desorption isotherm curves and 27Al MAS NMR spectroscopy, ammonia adsorption microcalorimetry and chemical analyses. The amount of adsorbed enzyme was found to be dependent on the Si/Al ratio of solid surfaces, pH of adsorption and, in the case of Al-containing material, on the ionic strength. Various mechanisms of adsorption are discussed. The activity of immobilized α-CT was examined using the transesterification of N-acetyl-L-phenylalanine ethyl ester with 1-propanol as test-reaction. The catalytic efficiency is strongly dependent on the nature of the support. In order to reduce the leaching of enzyme from the support surface and improve its reusability, cross-linking of the adsorbed enzyme was carried out by glutardialdehyde. In general, these mesoporous silica materials are attractive candidates for enzyme immobilization and biomedical applications due to their high surface area, tunable surface properties and pore size, large pore volume and biocompatibility.

α-Chymotrypsin catalyzed hydrolysis of p-nitrophenyl acetate in cationic microemulsions

The catalytic activity of α-chymotrypsin (α-CT) for the hydrolysis of p-nitrophenyl acetate has been studied in oil-in-water microemulsions formed by cetyltrimethylammonium bromide, n-butanol and alkanes (organic solvents). The specificity of α-chymotrypsin regarding the chain length of the organic solvent ( n-hexane, n-heptane and isooctane) in CTAB/ n-butanol microemulsions was tested in hydrolytic reactions of PNPA. The stability and activity of α-CT depend upon organic solvents. Under all the conditions employed, the reaction follows a Michaelis–Menten mechanism. The second order rate constant, k 2 was determined for α-CT catalyzed hydrolysis of p-nitrophenyl acetate at 27 °C. The chain length of oil influences the k cat and K M. The CTAB/ n-butanol/isooctane ( z = 1.07) system of o/w microemulsion shows the higher activity and stability of α-CT.

Catalytic activity of α-chymotrypsin in enzymatic peptide synthesis in ionic liquids

The catalytic activity of α-chymotrypsin in the enzymatic peptide synthesis of N-acetyl- l-tryptophan ethyl ester with glycyl glycinamide was examined in ionic liquids and organic solvents. The water content in 1-ethyl-3-methylimidazolium bis(fluorosulfonyl)imide ([emim][FSI]) affected the initial rates of peptide synthesis and hydrolysis. The activity of α-chymotrypsin was influenced by a kind of anions consisting of the same cation, [emim], when an ionic liquid was used as a solvent. The initial rate of peptide synthesis was improved 16-fold by changing from an organic solvent, acetonitrile, to an ionic liquid, [emim][FSI], at 25 °C. The activity of α-chymotrypsin in the peptide synthesis in [emim][FSI] was 17 times greater than that in acetonitrile at 60 °C, although the activity of α-chymotrypsin in the peptide synthesis gradually decreased with an increase in reaction temperature in [emim][FSI], similar to organic solvents. Moreover, α-chymotrypsin exhibited activity in [emim][FSI] and [emim][PF 6] at 80 °C.

Effects of polyethylene glycol on stability of α-chymotrypsin in aqueous ethanol solvent

The effects of polyethylene glycol (PEG) of different molecular weights (400, 2000, 6000, 12,000, 20,000, and 35,000) on the conformational stability and catalytic activity of α-chymotrypsin in 60% ethanol were studied. The inactivation caused by the organic solvent was not influenced by PEG 400. However, the PEGs with higher molecular weights up to 35,000 increased the stability of the enzyme, but this α-chymotrypsin stabilizing effect was molecular weight-independent. With increase of the molecular weight of PEG, a more stable tertiary structure of the enzyme was observed.

Effects of Ca 2+ on catalytic activity and conformation of trypsin and α-chymotrypsin in aqueous ethanol

The effects of calcium ions on the conformation and catalytic activity of trypsin and α-chymotrypsin were studied in aqueous ethanol. The activity of α-chymotrypsin was practically lost within 10 min in the presence of 60% ethanol while trypsin preserved about 40% of its original activity even in 85% ethanol at pH 3. The catalytic activity of α-chymotrypsin did not decrease in the presence of 1.2 M CaCl 2 and 0.6 M CaCl 2 with trypsin in ethanolic solvent. In the latter case an activation of enzyme was observed. The stabilizing effects of calcium ions were accompanied by an increase in the helical content in both enzymes, as followed by circular dichroism measurements.

Effects of polyhydroxy compounds on the structure and activity of α-chymotrypsin

The effects of glycerol, polyethylene glycol, fructose, glucose, sorbitol, and saccharose on the conformation and catalytic activity of α-chymotrypsin were studied in 0.1 M sodium phosphate buffer and buffered aqueous 60% ethanol (pH 8.0). The enzyme activity was practically completely lost within 10 min in 60% ethanol, but in the presence of stabilizers the activity was retained. With the exception of polyethylene glycol, the stabilizing effect decreased with increase of the incubation time. The preservation of the catalytic activity was accompanied by changes in the secondary and tertiary structures of α-chymotrypsin.

Increase of catalytic activity of α-chymotrypsin in organic solvent by co-lyophilization with cyclodextrins

α-Chymotrypsin was lyophilized in the presence of 2,3,6-tri-O-methyl β-cyclodextrin, and it displayed activity 40 fold higher than free α-chymotrypsin for transesterification in acetonitrile. α-Chymotrypsin which was co-lyophilized with hydroxypropylated β- or γ-cyclodextrins retained more than 98% of its initial activity after 6 h incubation in acetonitrile.

Study on tryptophan fluorescence and catalytic activity of α-chymotrypsin in aqueous-organic media

The fluorescence spectra of α-chymotrypsin were measured in aqueous-organic mixed solvents over a wide range of solvent composition. The tryptophan fluorophores were used as probes of the environmental polarity, and the structural changes of the enzyme were detected as changes of the difference in the emission wavelength between the enzyme and N-acetyl- l-tryptophan ethyl ester ( Δλ em). It was found that Δλ em correlated well with the catalytic activity of the enzyme, as expressed by the hydrolysis rate of N-acetyl- l-tyrosine ethyl ester. A linear relationship was established between the hydrolysis rate and Δλ em in aqueous acetonitrile, ethanol, THF, 1,4-dioxane, and DMF with high correlation coefficients.

Concentration- and Structure-Dependent Effects of Amides on Protease Activity in Organic Solvents

Abstract The catalytic activity of α-chymotrypsin (CT) in the transesterification of N-acetyl-l-tyrosine methyl ester to its ethyl ester in aqueous-organic media was markedly enhanced by replacing a part of water with formamide. The activity of CT was strongly dependent on the formamide/water ratio, and excess formamide retarded the activity. Addition of formamide to reaction mixtures at constant water contents exhibited similar activation–deactivation profiles for CT. A kinetic study revealed that the rate acceleration is due to an increase in kcat rather than a change in Km. At a given concentration of amides (0.5 M, M = mol dm−3), propionamide and DMF were much less effective than formamide for activation of CT. The results suggest that formamide interacts with CT in a different way from water.

Effects of Calcium Ion on the Catalytic Activity of α-Chymotrypsin in Organic Solvents

Addition of small amounts of calcium ion markedly accelerated the transesterification of N-acetyl-l-tyrosine methyl ester to its ethyl ester by the catalysis of α-chymotrypsin in organic solvents. Maximum increase of the reaction rate was about 12-fold in the presence of 25 μm of calcium ion in ethanol. The rate increase was strongly dependent on calcium ion concentration and nature of organic solvents. Esterification of N-acetyl-l-tyrosine and hydrolysis of N-acetyl-l-tyrosine ethyl ester by α-chymotrypsin in organic solvents were also accelerated by calcium ion. The reactions obeyed Michaelis–Menten kinetics, and the acceleration of the reactions was due to the increase in kcat.

Catalytic activity of -chymotrypsin in which histidine-57 has been methylated.

The properties of a derivative of alpha-chymotrypsin in which histidine-57 has been methylated have been examined. Although the modified enzyme binds substrate with the same affinity as does native alpha-chymotrypsin, acylation and deacylation occur at much decreased rates. As for native alpha-chymotrypsin, a basic group of pK(a) approx. 7 is involved in both acylation and deacylation. The significance of these results is considered in relation to the normal function of histidine-57.

The prototropic behavior of alpha-chymotrypsin and chymotrypsinogen in water and formaldehyde. An examination of the reactivity of the amino groups in formaldehyde.

1 A careful study of the ionic behavior of α-chymotrypsin and chymotrypsinogen has shown that all the α- and ɛ-amino groups of these proteins are capable of their normal ionization. 2 The ionic difference between chymotrypsinogen and α-chymotrypsin is that expected from the known route of activation and these may be attributed to two C-terminal carboxyl groups (pK′ 3.8 ± 0.2) and two N-terminal α-amino groups (pK′ 7.8 ± 0.2). 3 The two N-terminal amino groups react with formaldehyde to form hydroxymethyl derivatives with pK′≃ 5. 4 All the amino groups of the proteins examined react quantitatively with formaldehyde to form hydroxymethyl derivatives and this reaction appears to be complete in 15–30 sec. 5 α-Chymotrypsin is catalytically active in 3.1 M formaldehyde with: (a) diminution of the catalytic rate, (b) retention of all active sites, and (c) a bell-shaped kcat/Kmvs. pH profile similar to that obtained in water. These data coupled with the present findings appear to be inconsistent with the postulated involvement of the α-amino group of the N-terminal isoleucine residue in the catalytic activity of α-chymotrypsin.