Purification of ionic liquid stable Aspergillus assiutensis VS34 cellulase, and generation of nanobiocatalysts for potential biomass conversion to biofuel

Abstract

Abstract Ionic liquids (ILs) have gained immense attention as eco-friendly solvents for pretreatment of lignocellulosics for their potential bioconversion to biofuels, bio-chemicals, and other products. However, for saccharification of IL-pretreated biomass IL-stable cellulases are desired. IL-stable cellulase and xylanase enzyme preparation developed from a previously isolated Aspergillus assiutensis VS34 was used for saccharification of IL-pretreated biomass. Current study reports the purification of IL-stable cellulase (CMCase) from A. assiutensis VS34 based on salt precipitation and ion exchange chromatography. Functionality of the purified cellulase (2.10-fold) was observed by native-PAGE and zymography, and the molecular weight (27 kDa) was assayed by SDS-PAGE. Though optimum temperature and pH of CMCase was 50℃ and 6.0, respectively, but the enzyme showed considerable activity and stability over a wide range of temperature (40–80℃, 72–99%) and pH (3–11, 60–95%). The activity of enzymes was enhanced by certain metal ions (Ca2+, Cu2+, Mn2+, Mg2+, and Co2+), but decreased considerably with Hg2+. Km and Vmax of CMCase were 6.996 mg/ml and 16.103 µmol/min/mg, respectively. Process-apt properties of A. assiutensis VS34 CMCase reflect its application potential for a variety of processes including biomass conversion. Submerged fermentation was executed using A. assiutensis VS34 in wheat bran medium and a combined cellulase and xylanase preparation developed was acetone-concentrated. The nanobiocatalysts were prepared by immobilization of concentrated cellulase/xylanase enzyme preparation on magnetic nanoparticles (efficiency 78.04% and 58.44%, respectively), and were examined for their saccharification potential for IL-pretreated sugarcane bagasse for repeated cycles.