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

Author(s)

da Silva, Tony Marcio ^[1] ; Pessela, Benevides Costa ^[2] ; Rodrigues da Silva, Jean Carlos ^[3] ; Lima, Matheus Sanita ^[1] ; Jorge, Jodo Atilio ^[1] ; Guisan, Jose Manuel ^[4] ; Polizeli, Maria de Lourdes T. M. ^[1]

Affiliation

^[1] Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Biol, BR-14040901 Ribeirao Preto, SP - Brazil

^[2] CSIC, CIAL, Inst Invest Ciencias Alimentos, Dept Biotecnol & Microbiol Alimentos, E-28049 Madrid - Spain

^[3] Univ Sao Paulo, FMRP, Dept Bioquim & Imunol, BR-14040901 Ribeirao Preto, SP - Brazil

^[4] CSIC, Inst Catalisis, Dept Biocatalisis, E-28049 Madrid - Spain

• Document type: Journal article
• Source: JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC; v. 104, p. 95-100, JUN 2014.
• Web of Science Citations: 8

Abstract

A beta-glucosidase from Aspergillus japonicus was produced on submerged fermentation using sugar cane bagasse in nature as carbon source, and incubated at 30 degrees C, for 72 h. After that, the dialyzed crude extract containing the active beta-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 beta-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 beta-glucosidase presented a half-life of 20 min, at 60 degrees C, while the MANAE-agarose and DEAE-cellulose derivatives presented a half-life of 25 and 48 h, respectively. The optima pH for soluble P-glucosidase, MANAE- and DEAE derivatives was 4.5. The optimal temperature for DEAE derivative and soluble enzyme was 60 degrees C and for MANAE-agarose derivative was 65 degrees C. The K-m values for soluble enzyme, MANAE-agarose and DEAE-cellulose derivatives using beta-pNPG as substrate were 1.4, 2.6 and 2.0 mg/mL, respectively. Using cellobiose as substrate the K-m values were 2.8, 3.4 and 3.7 mg/mL, respectively. The V-max values using beta-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 V-max 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 pNP beta-D-glucopyranoside. (C) 2014 Elsevier B.V. All rights reserved. (AU)