α-chymotrypsin superactivity in cetyltrialkylammonium bromide-rich media |
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Authors: | Francesco Alfani Maria Cantarella Nicoletta Spreti Raimondo Germani Gianfranco Savelli |
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Institution: | (1) Department of Chemistry, Chemical Engineering and Materials, University of L'Aquila, 67040 L'Aquila, Italy;(2) Department of Chemistry, University of Perugia, 06123 Perugia, Italy |
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Abstract: | α-Chymotrypsin (α-CT) activity was tested with N-glutaryl-l-phenylalanine p-nitroanilide in buffered media with added cationic surfactants. The effect of the commercial cetyltrimethylammonium
bromide (CTABr) was compared with that of three other surfactants with ethyl (CTEABr), propyl (CTPABr), and butyl (CTBABr)
head groups. These were synthesized and purified in this laboratory. Surfactant head groups provided distinct environments
that largely modulated the catalytic performance. Larger alkyl head group hydrophobicity led to a marked enhancement of α-CT
activity. CTBABr-rich media induced the highest superactivity.
Kinetic measurements were performed in Tris-HCl buffer at a surfactant concentration either below or above CMC, and α-CT superactivity
occurred in both media. Positive interactions between the enzyme and surfactants happened independently of thesupramolecular
organization of the medium. The reaction followed the Michaelis-Menten kinetics. The substrate to micelle aggregates binding
constant was used to calculate the substrate concentration available for catalysis. The k
cat to k
m ratio was in CTBABr-rich media always higher than in pure buffer and depended on the surfactant concentration. α-CT superactivity
depended on the pH value of buffer solution. Enzyme inactivation followed a single-step mechanism in pure buffer and a series
mechanism in the presence of a surfactant. The rate of activity decay obeyed a first-order kinetics. |
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Keywords: | α -Chymotrypsin superactivity stability cationic surfactants micellar aggregates |
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