Improved Activity and Kinetics of Endoglucanase Biofuel Enzyme with Addition of an AzoTAB Surfactant

Abstract

Endoglucanases degradeβ-1,4-glycosidic bonds of crystalline cellulose into insoluble or soluble cellooligosaccharides at the solid-liquid interface. The enhanced activity and kinetics of endoglucanase fromAspergillus nigerwith addition of a light responsive azobenzene trimethyl ammonium bromide (azoTAB) surfactant is studied. AzoTAB is a photoresponsive surfactant that exists as a relatively-hydrophobictransisomer under visible light (434 nm) and a relatively-hydrophiliccisisomer under UV light (350 nm). Endoglucanase catalytic activity can be controlled with light illumination slightly for microcrystalline cellulose natural substate (∼15%) and significantly for p-nitrophenol-based model substrate (∼2-fold) by switching between thetrans(higher enzyme binding affinity, resulting higher enzyme unfolding) andcisform of azoTAB. Endoglucanase activity increases 45% towards avicel crystalline substate and 4-fold towards 4-nitrophenyl β-D-cellobioside substrate in the presence of 0.4 mM azoTAB under UV light (90%cisand 10%transisomers). In comparison, endoglucanase catalytic activity increases 5-10% towards crystalline cellulose substrate with the addition of sodium dodecyl benzene sulfonate (SDBS), sodium dodecyl sulfate (SDS) and dodecyl trimethyl ammonium (DTAB). 0.4 mM AzoTAB-UV addition leads to an increase of maximum endoglucanase adsorption (Emax) from 7.89 mg enzyme/g avicel to 12.92 mg enzyme/g avicel and catalytic enzyme efficiency (kcat/KM) from 0.031 L/(mg.s) to 0.061 L/(mg.s). It is found that adsorbed enzyme concentration on substrate correlates to enzyme specific activity for azoTAB containing reaction. Additionally, 40-50% activity enhancement and increased bound enzyme to substrate are detected with azoTAB addition at different enzyme, substrate, and inhibitor concentrations. Improvement of substrate properties of azoTAB addition is associated with lower reciprocal terms of Michaelis constant (KM), the adsorption coefficient (Kad) and fractal parameter (h) values similar to the presence of other surfactants in this study and the literature. Furthermore, 45-50% activity enhancement via azoTAB surfactant preserved for all three cellulase enzyme mixture of endoglucanase, cellohydrolase andβ-glucosidase. Consequently, azoTAB can be applied as profitable additive for the heterogenous enzymatic cellulose hydrolysis, resulting in a 30% decrease in the enzyme load based on the specific activity, adsorption, and fractal kinetics results.