Immobilization and characterization of pancreatic lipase enzyme on composite carriers

Lipases (triacylglycerol hydrolases, EC 3.1.1.3) are the enzymes which catalyze the hydrolysis or synthesis of fats and oils. In addition to biochemical and physiological properties of lipases, they become subject of research due to industrial applications. Lipases are commonly found enzymes which hydrolyze solid-liquid oils to free fatty acids, diacylgliserols, monoaçilgliseroller and glycerol. They also catalyze reactions like esterification, transesterification, aminolysis in the organic solvents. Because of a wide variety of reactions they can be use in the separation of racemic mixtures, synthesis of new surfactants and pharmaceuticals, oil transformations and detergent manufacturing. Given the economic point of view, the use of lipase in industrial scale, an immobilization is required. Immobilized enzymes are preferred to natural enzymes because they can be reused for many times, and for a long time, they can be applied to continuous operations, they are stabile and resistant to environmental conditions (pH, temperature, etc.). In this study, porcine pancreatic lipase enzyme was immobilized on composite carries which was prepared by economical and inexpensive biopolymer such as chitosan. Chitosan is widely used in laboratory-scale studies for immobilization of enzymes although there are some obstacles for industrial applications. Cross-linked chitosan beads have greater mechanical strength, but they still have problems with operational applications. Density of the beads is very close to water and its texture is very soft. In this study, chitosan mixed with carbon nanotube (Multiwalled carbon nanotube, MWCNTs) and perlite for increasing the mechanical strength than the suspension dropped in tripolyphosphate solution. Characterization of composite beads was done by fourier transform infrared (FTIR) spectroscopy, scanning Electron microscopy (SEM) and the thermal gravimetric analysis (TGA). To optimize the immobilization conditions, effect of crosslinking time, enzyme immobilization time and effect of lipase concentration were examined. Optimum ECH crosslinking time was found as 4 hours and 17 hours for chitosan-perlite and chitosan-MWCNTs respectively. Optimum enzyme immobilizasyon time was found as 24 hours for chitosan-perlite and chitosan-MWCNTS composite beads. Optimum enzyme concentration was determined as 2 mg/ml for both of them. In addition, effect of pH and temperature on the activity of free and immobilized lipase was examined. The optimum pH of the immobilized lipase was shifted slightly to the alkaline region relative to the free enzyme and it was found as pH 8.0 for both carriers. Optimum temperature of free lipase was reported as 40 ˚C and it was found as 50 ˚C for the enzyme immobilized on both chitosan-perlite and chitosan-MWCNTs. Also, thermal, pH and storge stability of free and immobilized enzyme was compared and the reusability of immobilized lipase was examined. The thermal stability of immobilized lipase was better than that of the free lipase. The immobilized enzyme was more stable than its free form in this pH range. For the developed method, immobilization lipase was showed greater storage stability than free enzyme. At the end of 40 days, while free lipase had 20% remaning activity, this value was calculated as 59 % and 63 % for lipase immobilized on chitosan-perlite and chitosan-MWCNTs composite beads respectively at +4 0C. At the end of same period of storage, free lipase had 5 % remaning activity and this value was calculated as 48 % and 55 % for lipase respectively at room temperature. When reusability of immobilized lipase was examined, it retained 50 % of its original activity after 15 and 13 reuses when lipase immobilized on chitosan-perlite and chitosan-MWCNTs composite beads respectively.