Kinetic characterization of lipase from thermomyces lanuginosus and its use in the production of biodiesel from sunflower oil

Abstract

Cilj ovoga rada bio je razviti kinetički model hidrolize p-NPP-a katalizirane lipazom koristeći tri različita otapala (acetonitril, metanol i etanol), procijeniti kinetičke parametre procesa (Vm i Km), istražiti utjecaj koncentracije enzima, vrste otapala i temperature na aktivnost lipaze te provesti jednostupanjsku i četverostupanjsku reakciju enzimske sinteze biodizela iz jestivog suncokretovog ulja. Mjerenjima početne reakcijske brzine pokazano je da su najvede volumne aktivnosti lipaze postignute koristeći 5 % (g/g) koncentraciju enzima u pokusima sa svim ispitanim otapalima (Vm,etanol = 1373,852 U/mL, Vm,acetonitril = 318,817 U/mL, Vm,metanol = 1141,068 U/mL). Najveći afinitet prema supstratu (p-NPP) lipaza pokazuje u sustavu s acetonitrilom kao otapalom (Km = 1,0217 mM). U jednostupanjskim i četverostupanjskim procesima proizvodnje biodizela iz jestivog suncokretovog ulja pri različitim temperaturama (T = 40 – 60 °C), najviši udio metil estera postignut je pri T = 40 °C. U jednostupanjskoj reakciji udio metil estera iznosi 94,9 % nakon 96 sati, dok je za isto vrijeme u četverostupanjskoj reakciji udio metil estera iznosio 89,0 %.The aim of this study was to develop a kinetic model for hydrolysis of p-NPP catalyzed with lipase using three different solvents (acetonitrile, methanol and ethanol), to evaluate the kinetic parameters (Vm and Km), to investigate the influence of enzyme concentration, solvent type and temperature on lipase activity and to perform single step and four step reaction of biodiesel synthesis from edible sunflower oil catalyze by lipase. The initial reaction rate measurements shown that the highest volume activity of lipase was achieved using 5% (g/g) concentration of enzyme in the assays with all tested solvents (Vm, ethanol = 1373.852 U/mL, Vm, acetonitrile = 318.817 U/mL, Vm,methanol = 1141.068 U/mL). The highest affinity to the substrate (p-NPP) lipase shows in reaction with acetonitrile (Km = 1.0217 mM). In the single step and four step process of biodiesel production from edible sunflower oil at different temperatures (T = 40 – 60 °C), the highest fatty acids methyl esters share were achieved at T = 40 °C. 94.9 % fatty acids methyl esters share was achieved in single step reaction after 96 h and 88.9 % in four step reaction for the same duration