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 °C and pH 4. EGLB was stable for 3 h at temperatures below 60 °C, with more than 90% of its activity remaining. The enzyme was specific for substrates with β-1,3 and β-1,4 linkages. In Lineweaver-Burk plot analysis, the K(m) and V(max) values of EGLB for β-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.
Department of Bioengineering, Tatung University, 40 Chung-Shang North Road, 3rd Section, Taipei 104, Taiwan.
Chien-Huang
CH
Li
Hsing-Ren
HR
Wang
Tsong-Rong
TR
Yan
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004795:Enzyme Stability; D015964:Gene Expression Regulation, Bacterial; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008670:Metals; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020218:Response Elements; D013379:Substrate Specificity
D007477:Ions; D008670:Metals; D011994:Recombinant Proteins; D002480:Cellulase