Immobilization and high stability of an extracellular β-glucosidase from Aspergillus japonicus by ionic interactions

A β-glucosidase from Aspergillus japonicus was produced on submerged fermentation using sugar cane bagasse in nature as carbon source, and incubated at 30 °C, for 72 h. After that, the dialyzed crude extract containing the active β-glucosidase was purified from three successive steps using DEAE-fractogel, MANAE-agarose and octyl-sepharose chromatographic columns. The enzyme migrated as a single band on SDS-PAGE and its molecular weight was estimated to be 114 kDa. The amino acid sequence determined by mass spectrometry demonstrated a similar structure for the β-glucosidase from Aspergillus niger and A. kawachii. The purified enzyme was immobilized by entrapment with sodium alginate beads; covalent attachment using activated CNBr-agarose and ionic interaction on MANAE-agarose and DEAE-cellulose. Soluble β-glucosidase presented a half-life of 20 min, at 60°C, while the MANAE-agarose and DEAE-cellulose derivatives presented a half-life of 25 and 48 h, respectively. The optima pH for soluble β-glucosidase, MANAE- and DEAE derivatives was 4.5. The optimal temperature for DEAE derivative and soluble enzyme was 60°C and for MANAE-agarose derivative was 65°C. The Km values for soluble enzyme, MANAE-agarose and DEAE-cellulose derivatives using β-pNPG as substrate were 1.4, 2.6 and 2.0 mg/mL, respectively. Using cellobiose as substrate the Km values were 2.8, 3.4 and 3.7 mg/mL, respectively. The Vmax values using β-pNPG were 24, 25.8 and 13.5 U/mg proteins for the soluble enzyme, MANAE-agarose and DEAE-cellulose derivatives, respectively. Using cellobiose as substrate the Vmax values were 146, 152 and 100.5 U/mg protein. The MANAE and DEAE derivatives presented a good operational stability being evidenced 50 and 60% residual activity after 5 cycles of reaction with the substrate pNPβ-d-glucopyranoside.This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (process n° 2010/52322-3) and Conselho de Desenvolvimento Científico e Tecnológico (CNPq, process n° 563260/2010-6). Tony Marcio da Silva was a recipient of a FAPESP Fellowship and this work was part of his Pos Doctoral studies. This project is part of National Institute of Science and Technology of the Bioethanol.Peer reviewe