Ethylene glycol and the thermostability of trypsin in a reverse micelle system

Abstract

The influence of ethylene glycol (EG) on the kinetics of hydrolysis of N-alpha-benzoyl-L-arginine ethyl ether catalyzed by trypsin encapsulated in sodium bis-(2-ethylhexyl)sulfosuccinate (AOT)-based reverse micelles was studied at different temperatures. Ethylene glycol was shown to shift the range of the trypsin activity in the reverse micelles towards higher temperatures. Infrared spectroscopy showed a stabilizing effect of EG on the secondary structure of the protein in the system of reverse micelles. Electron spin resonance spectroscopy showed that the solubilized protein affected the interactions of EG with the polar head groups of AOT and altered the rigidity of the micellar matrix. The results indicate that EG increases the thermostability of the solubilized enzyme in microemulsion media by two mechanisms.

The effect of dynamic high-pressure microfluidization on the activity, stability and conformation of trypsin

“…The improvement of thermal stability after DHPM treatment might be due to the alteration of trypsin into a stable hydrated structure. The alteration of trypsin is caused by the dehydration of hydrogen-bonded water (de Jongh, Goormaghtigh, & Ruysschaert, 1997;Stupishina, Khamidullin, Vylegzhanina, Faizullin, & Zuev, 2006).…”

Comparative evaluation of bioactivity of crystalline trypsin for drying by Fourier-transformed infrared spectroscopy

“…The activity decreased as the temperature increased during heat-treatment, by 11% at 100 • C, 18.5% at 125 • C, 49.9% at 150 • C, and 99.3% at 180 • C. The loss of enzymatic activity was smaller at low temperature, but larger at high temperature. The activity of heat-treated trypsin in a solid-state was much greater than that in solution [14]. Fig.…”

“…The result suggested that the ␤-sheet was denatured by the heat-treatment, meaning that the free carbonyl groups and vapor water might be increased by dehydration of the ␤-sheet (hydrated carbonyl groups). Stupishina et al [14] and de Jongh et al [15] reported the effect of heat-treatment on the enzymatic reaction kinetics of trypsin in solution. They identified structural changes by FT-IR, and concluded that the inter-and intra-molecular hydrogen bonds of trypsin might be reduced with an increase in the temperature due to the dehydration of hydrogenbonded water.…”

“…The spectra were pre-treated by area normalization to correct spectral fluctuations due to particle size. Stupishina et al [14] assigned major bands of trypsin as follows: those at 1634, 1650, 1655, and 1660 cm −1 were associated with C O due to a ␤-sheet, other structure, helix, and ␤-turn, respectively. Winters et al [13] also reported that the bands at 1636, 1660, and 1687 cm −1 of trypsin associated with C O due to a ␤-sheet, ␣-helix, and ␤-turn, respectively.…”

“…The secondary structural change of trypsin in solution on heattreatment was examined qualitatively using IR spectroscopy by Stupishina et al [14]. They suggested that the kinetic rate constant depended on the change of secondary structure with temperature in micelles of trypsin.…”