Optimization of esterification activity of lipase from Candida rugosa immobilized using microwave irradiation

Abstract

Lipases are very efficient biocatalysts with wide application in synthesis of important ingredients of food, cosmetics and pharmaceutical products, due to their capacity to catalyze both, ester synthesis and ester hydrolysis. The preparation of stable and active immobilized derivatives of lipases is necessity for their application in industrial enzymatic processes. In this work, the optimization of lipase from C. rugosa immobilization by microwave irradiation was performed, since it was previously reported that immobilization process can be drastically accelerated by means of microwave irradiation, even resulting with slight increase of lipase activity. Eupergit®, commercial support with active epoxy groups, was used as immobilization support. In first stage of our study, the immobilization time and ionic strength of immobilization buffer were optimized. It was found out that the highest immobilized activity can be achieved at high ionic strengths (1 M buffer) after 3 min, while further increase of immobilization time led to decrease of lipase activity. Then, the immobilized derivative obtained at optimum conditions was applied in synthesis of amyl isobutyrate in organic solvent. Key reaction factors (temperature, water concentration, immobilized lipase concentration, and substrate molar ratio) were optimized using response surface methodology. The substrate conversion higher above 85% was achieved in our study. The statistical analysis revealed that each of analyzed factors had significant effect on yield of ester, with initial enzyme concentration and substrate molar ratio being the most prominent factors. The second-order regression model that describes the effect of all four factors on substrate conversion was established. The optimum values of factors were: temperature 50°C, initial immobilized enzyme concentration 220 mg ml-1, added water concentration 0.1% (v/v), and molar ratio acid/alcohol 2.5.

Lipaze su enzimi koji imaju široku primenu u prehrambenoj, kozmetičkoj i farmaceutskoj industriji. U industrijskoj praksi primenjuju se imobilisane lipaze jer se imobilizacijom povećava njihova stabilnost i ekonomičnost enzimskih postupaka. U ovom radu optimizovana je imobilizacija lipaze iz C. rugosa na komercijalni nosač Eupergit® pomoću mikrotalasnog zračenja. Vreme izlaganja mikrotalasima i molaritet korišćenog pufera optimizovani su sa ciljem dobijanja imobilizata maksimalne aktivnosti. Radi optimizovanja reakcije sinteze amil-izobutirata, pomoću centralnog kompozitnog rotatabilnog plana ispitan je uticaj četiri reakciona faktora. Ustanovljeno je da su optimalni uslovi sinteze estra temperatura 50°C, početna koncentracija imobilisanog enzima 220 mg ml-1, koncentracija dodate vode 0,1% i molarni odnos supstrata 2,5.