Kinetic modeling of cellobiose by a β-glucosidase from Aspergillus fumigatus

Abstract

The final step in lignocellulose enzymatic saccharification is the cellobiose conversion toglucose by β-glucosidases (BG). In this work, a valid kinetic model to describe cellobiosedegradation for an industrial mixture of BG enzymes present in Aspergillus fumigatus isselected. Firstly, the enzyme mixture was characterised in terms of protein content andenzymatic activity on p-NPG (1326 U mLpreparation−1), determining the molecular weight ofthe only BG activity band observed in zymograms by SDS-PAGE and MALDI-TOF: 94 kDa.Subsequently, to select the correct kinetic model for the enzymatic hydrolysis of cellobiose,a combined strategy was performed: Firstly, non-linear regressions were applied to initialhydrolysis rate data for different enzyme concentrations and initial substrate and productconcentrations, observing inhibition by cellobiose and glucose. Secondly, the optimal kineticmodel was discriminated by a coupled non-linear regression-DOE numerical integrationapproach, by fitting several possible kinetic models involving different product inhibitionmechanisms to progress curve data from runs at various initial substrate concentrationsand temperatures. The best kinetic model involves non-competitive substrate inhibitionand product competitive inhibition with two binding sites for glucose.