1. Enzymatic hydrolysis of N-benzoyl-L-tyrosine p-nitroanilide by α-chymotrypsin in DMSO-water/AOT/n-heptane reverse micelles. A unique interfacial effect on the enzymatic activity
Fernando Moyano, Evangelina Setien, Juana J Silber, N Mariano Correa Langmuir. 2013 Jul 2;29(26):8245-54. doi: 10.1021/la401103q. Epub 2013 Jun 20.
The reverse micelle (RM) media are very good as nanoreactors because they can create a unique microenvironment for carrying out a variety of chemical and biochemical reactions. The aim of the present work is to determine the influence of different water-dimethyl sulfoxide (DMSO) mixtures encapsulated in 1,4-bis-2-ethylhexylsulfosuccinate (AOT)/n-heptane RMs on the enzymatic hydrolysis of N-benzoyl-L-tyrosine p-nitroanilide (Bz-Try-pNA) by α-chymotrypsin (α-CT). The reaction was first studied in homogeneous media at different DMSO-water mixture compositions and in DMSO-water/AOT/n-heptane RMs. The hydrolysis rates of Bz-Try-pNA catalyzed by α-CT were determined by UV-vis spectroscopy. The reaction follows the Michaelis-Menten mechanism and the kinetic parameters: kcat, KM, and kcat/KM were evaluated under different conditions. In this homogeneous media, DMSO plays an important role in the solubilization process of the peptide which is almost insoluble in water, but it has a tremendous impact on the inactivation of α-CT. It is shown that the enzyme dissolved in a 20% molar ratio of the DMSO-water mixture does not present enzymatic activity. Dynamic light scattering has been used to assess the formation of DMSO-water/AOT/heptane RMs at different DMSO compositions. The results also show that there is preferential solvation of the AOT RM interface by water molecules. To test the use of these RMs as nanoreactors, the kinetic parameters for the enzymatic reaction in these systems have been evaluated. The parameters were determined at fixed W(S) {W(S) = ([water] + [DMSO])/[AOT] = 20} at different DMSO-water compositions. The results show that the Michaelis-Menten mechanism is valid for α-CT in all the RM systems studied and that the reaction takes place at the RM interface. Surprisingly, it was observed that the enzyme encapsulated by the RMs show catalytic effects with similar kcat/KM values at any DMSO composition investigated, which evidence that DMSO molecules are localized far from the RM interface.
2. Proteinase activity in potato plants
K Santarius, H D Belitz Planta. 1978 Jan;141(2):145-53. doi: 10.1007/BF00387881.
Several vegetative tissues of potato plants were screened for proteinase activity. Both endopeptidase and exopeptidase activities were investigated using gelatin and L-amino acid-4-nitroanilides (benzoyl-L-arginine-4-nitroanilide/BAPA, glutaryl-L-phenyl-alanine-4-nitroanilide/GLUPHEPA, alanine-4-nitro-anilide/APA, leucine-4-nitroanilide/LPA, and benzoyl-L-tyrosine-4-nitroanilide/BTPA) as substrates. Leaves and rootes were found to contain the highest levels of endopeptidase activity; lesser activities were detected in flower petals, sprouts, and tubers. Three different types of proteinases, L-BAPAase (serine proteinase), APAase (thiol proteinase), and BTPAase (sensitive to reducing agents), were characterized in various physical and chemical properties. Their temperature optima were determined to be 25° (L-BAPAase) and 40° (BTPAase, APAase) respectively; their pH optimum was between 8.6 and 9.0, their isoelectric points were between pH 4.25 and 6.0, and their molecular weight was estimated 70,000 (L-BAPAase, APAase) and between 150,000-250,000 (BTPAase). The trypsin-like activity against L-BAPA was inhibited by diisopropylfluorophosphate and by tosyllysine-chloromethyl ketone, but not by trypsin inhibitors from potato and legume.
