Enhancement lipase activity via immobilization onto chitosan beads used as seed particles during fluidized bed drying: Application in butyl butyrate production

We present a method for lipase immobilization via fluidized bed technology. The enzyme drying/immobilization solution consisted of phosphate buffer (50 mM pH 7.0) containing the lipase (5.0 mg(protein)/g(chitosan)), 200 mM of NaCl and PEG-1500 (1.5 % m/v). The lipase immobilized on glutaraldehyde-activated enhanced 46.5 % of initial lipase activity. The optimum pH for the free lipase was slightly acid (pH 6.5), whereas the optimum pH for the immobilized systems shifted to the alkaline region (pH 8.0). As for the optimum temperature, it went from 45 degrees C for the free lipase to 50 degrees C for the immobilized systems. The immobilized systems had half-life of 5.81 h and stabilization factor of 10.75 at 80 degrees C. The kinetic data showed that the lipase immobilized on chitosan beads activated with glutaraldehyde was catalytically more efficient (K-M: 0.015, V-max: 170.4 mu mol/min/mg, and V-max/K-M: 11,360) than the free lipase (K-M: 0.035, V-max: 120.3 mu mol/min/mg, and V-max/K-M: 3437); displayed low moisture content and water activity (moisture: 3.84 % and A(W): 0.10); and retained 85.5 % of its initial activity (vs 16.5 % retained activity for the free lipase) after storage for six months. After ten reuse cycles, said immobilized system retained 75.2 % of its initial activity. We employed this system as biocatalyst to esterify butyric acid with butanol. We developed a centered faced central composite design 22 and a mathematical model to describe how the ester content behaved as a function of temperature and amount of biocatalyst, as independent variables. Under optimized conditions (temperature of 45 degrees C and biocatalyst mass of 0.5 g), 18 g of butyl butyrate/L was attained within 6 h, which corresponded to an esterification yield of 98.96 %.

Automated summary

The method of lipase immobilization via fluidized bed technology led to an increase in enzyme activity and a shift towards alkaline conditions for optimal activity. The immobilized systems demonstrated longer half-life and stability factor at high temperatures.