Preparation of castor oil porous organosilica composites for immobilization of trypsin enzyme

Porous organosilica composites have been prepared by co-condensation of 3- mercaptopropyltriethoxysilane and tetraethylorthosilicate in a 1:4 molar ratio respectively using castor oil and dodecylamine templates. FTIR spectroscopy was done to confirm the presence of tethered functional groups on the surface of mesoporous materials before immobilizing the enzyme. Trypsin enzyme was then covalently immobilized on the thiol modified porous materials by Thiol-disulflde interchange. Nitrogen physisorption studies showed that the prepared porous materials have surface areas ranging from 357 to 605 m /g with pore diameters ranged from 5 to 20 nm as determined by BJH method. The average pore volume of thiol modified micelle templated silica materials was 1.0336 cm /g and decreased to 0.8169 cm3/g upon trypsin immobilization. Both free and immobilized trypsin showed maximum activity on N-a- benzoyl-D, L-arginine-/?-nitroanilide hydrolysis at a temperature range of 35 to 38 °C. Covalently immobilized trypsin were stable up to 55 °C while free trypsin had a sharp decrease on activity at that temperature. The activation energies of N-a- benzoyl-D, L-arginine-p-nitroanilide hydrolysis for free and immobilized trypsin were 47.8 and 41.6 kJ /mol respectively, whereas Arrhenius factors were 33 and 23 U /g protein. The Vmax for free and covalently immobilized trypsin on micelle templated silica prepared by castor oil and dodecylamine were 0.32, 0.182 and 0.011 U, respectively whereas KM values were 3.8 xlO'04, 5.2 xlO’04 and 3.1x10‘04 M, respectively. The immobilized trypsin was capable of being reused up to four cycles with slight decrease on its activity.