Carrier-free Immobilized Porcine Pancreatic Lipase

Cross-linked enzyme aggregates（CLEAs）, a carrier-free immobilized method throughchemical cross-linking the physical aggregates,has numerous advantages such as easypreparation, strong stability, high repeated utilization rate, low preparing cost and highenzyme activity recovery and has recently attract the attention of enzymology workers.Porcine pancreatic lipase(EC3.1.1.3) is an important enzyme of hydrolysis, Which couldcatalyze the hydrolysis of triglyceride to glycerol and fatty-acids, plays an important role inthe hydrolysis or formation of lipids. The application of CLEAs to porcine pancreatic lipaseexpectedly improves the stability and enzyme activity recovery dramatically.But up to now,the CLEAs of porcine pancreatic lipase has barely been reported.The hydrolysis condition of porcine pancreatic lipase was optimized, and deeplyinvestigated the preparation and properties of cross-linked porcine pancreatic lipaseaggregates. Then the cross-linked porcine pancreatic lipase were used to catalyze thesynthesis of bio-diesel. Major contents were presented as follows:The conditions such as temperature, pH, reaction time, concentrations of substrate andenzyme could affect the activity of porcine pancreatic lipase. Under the conditions of pH7.0,substrate concentration of1.0mg/mL and enzyme concentration of0.3mg/mL at45oC for20min, the porcine pancreatic lipase activity was the highest.Out of ammonium sulfate, ethanol, acetone and polyethylene glycol, ammonium andacetone present best ability to precipitate porcine pancreatic lipase. The enzyme wascompletely precipitated with no loss of activity in the present of50%(V/V) acetoneconcentration and60%saturation of ammonium sulfate. Addition of bovine serumalbumin(BSA) as a protein feeder before precipitation effectively solved the problem ofineffective cross-linking when porcine pancreatic lipase directly cross-linked withglutaraldehyde. After optimization, when the mass ratio between bovine serum albumin(BSA)and enzyme was1:10, up to90%of the starting enzyme activity maintained in thecross-linked porcine pancreatic lipase aggregates.The stability and acitivity of procinepancreatic lipase enhanced after carrier-free immobilization.Precipitating enzyme by the use of ammonium sulfate of60%saturation and acetone of50%(V/V) concentration,the cross-linking by glutaraldehyde to prepare cross-linked enzyme aggregates，The optimum paraters of preparation are：enzyme concentration of0.3mg/mL,precpitation time for1.0h, addition of1%(V/V) glutaraldehyde are3mL and9mLrespectively,cross-linking time for2.0h,the cross-linked enzyme aggregates activity reachedto1433U/g under such conditions. Compared to free porcine pancreatic lipase, cross-linkedporcine pancreatic lipase aggregates had enhanced pH, temperature and organic reagentstolerance. Exhibited higher thermostability and lower thermal inactivation rate under hightemperature and the pH stability broadened from6.5~7.5to6.0~9.0; Moreover, cross-linkedporcine pancreatic lipase retained60%of its starting enzyme activity after incubated at4oCrefrigerator for120dates, while the free enzyme deactivate completely; Methanol reduceddrastically the catalytic activity of cross-linked porcine pancreatic lipase aggregates and freeporcine pancreatic lipase after72h of incubation, residual activity of immobilized enzymewas around70%of the initial activity, however, free enzyme just retained40%of the initialactivity.The conditions of porcine pancreatic lipase and cross-linked porcine pancreatic lipasecatalyzed synthesis of bio-diesel was optimized. Under the condition of the methanol to oilmolar ratio5:1, enzyme concentration of5%at35oC for15h, the free enzyme gained thebest conversion of75%, compared to85%conversion of cross-linked enzyme aggregatesunder the conditon of methanol to oil molar ratio4:1,enzyme concentration of4%at40oC for12h.