Laccase Validation as Pretreatment of Agave Waste Prior to Saccharification: Free and Immobilized in Superparamagnetic Nanoparticles Enzyme Preparations

Abstract

In the present study free and immobilized on magnetic nanoparticles coated with chitosan (C-MNP) laccase was applied to validate their effects on agave biomass waste as pretreatment before enzymatic saccharification. C-MNP were prepared by one step coprecipitation. Trametes versicolor laccase was immobilized via glutaraldehyde reaction. Fourier-Transformed Infrared spectra, magnetization measurements, and high resolution transmission electron microscope were performed to characterize bio-nanocomposite. Operational properties of free and immobilized laccase were evaluated spectrophotometrically using catechol as substrate. Effects of free and immobilized laccase on Agave atrovirens biomass waste, pretreated previously by autoclaving, were valued by glucose release after cellulase catalyzed hydrolysis. Immobilization technique yielded 82% of protein immobilization, and 89% of initial activity were retained. Immobilized enzyme showed better thermal and storage stability than free enzyme; Km value increased only at 1.25 times, and its activity behavior as pH and temperature function was maintained. Immobilized laccase retained 50% of its activity after 5 cycles. Free laccase applied for 12 h at 3 U (solid vegetal material g−1) in cactus Agave atrovirens biomass led to raise the yield of enzymatic hydrolysis (with Celluclast at 15 U g−1) at 11%, while immobilized laccase led to decrease yield at 16%. It was demonstrated that the interaction between bio-nanocomposite and lignocellulosic material probably hindered magnetic separation of immobilized enzyme. Thus, free laccase may be applied as agave biomass treatment before enzymatic hydrolysis. Immobilized enzyme may be applied for other biotechnological processes where magnetic separation may be useful for development of new technologies.