Cloning, Purification, and Characterization of a Heat- and Alkaline-Stable Endoglucanase B from Aspergillus niger BCRC31494

Endoglucanase B (EGLB) derived from Aspergillus niger BCRC31494 has been used in the food fermentation industry because of its thermal and alkaline tolerance. It was cloned and expressed in Pichia pastoris. According to sequence analysis, the gene open reading frame comprises 1,217 bp with five introns (GenBank GQ292753). According to sequence and protein domain analyses, EGLB was assigned to glycosyl hydrolase family 5 of the cellulase superfamily. Several binding sites were found in the promoter region. The purified recombinant enzyme was induced by 0.5% methanol, and it exhibited optimal activity at 70 degrees C and pH 4. EGLB was stable for 3 h at temperatures below 60 degrees C, with more than 90% of its activity remaining. The enzyme was specific for substrates with beta-1,3 and beta-1,4 linkages. In Lineweaver-Burk plot analysis, the K-m and V-max values of EGLB for beta-D-glucan were 134 mg/mL and 4.68 U/min/mg, respectively. The enzyme activity was increased by 1.86-fold by Co2+ and by 2-fold by Triton X-100 and Tween 80. These favorable properties make EGLB a potential candidate for use in laundry and textile industrial applications.