title: Characteristics of bifunctional acidic endoglucanase (Cel5B) from Gloeophyllum trabeum.
abstract: The endoglucanase (Cel5B) from the filamentous fungus Gloeophyllum trabeum was cloned and expressed without a signal peptide, and alanine residue 22 converted to glutamine in Pichia pastoris GS115. The DNA sequence of Cel5B had an open reading frame of 1,077 bp, encoding a protein of 359 amino acid residues with a molecular weight of 47 kDa. On the basis of sequence similarity, Cel5B displayed active site residues at Glu-175 and Glu-287. Both residues lost full hydrolytic activity when replaced with alanine through point mutation. The purified recombinant Cel5B showed very high specific activity, about 80- to 1,000-fold and 13- to 70-fold in comparison with other endoglucanases and cellobiohydrolase, on carboxymethylcellulose and filter paper, respectively, at pH 3.5 and 55°C. Cel5B displayed bifunctional characteristics under acidic conditions. The kinetic properties of the enzyme determined using a Lineweaver-Burk plot indicated that Cel5B is a catalytically efficient cellulolytic enzyme. These results suggest that Cel5B has high bifunctional endo- and exoglucanase activity under acidic conditions and is a good candidate for bioconversion of lignocellulose.
authors.affiliation: Department of Forest Products and Technology, Chonnam National University, Gwangju, 500-757, Republic of Korea.
authors.forename: Ho Myeong
authors.initials: HM
authors.lastname: Kim
authors.forename: Yoon Gyo
authors.initials: YG
authors.lastname: Lee
authors.forename: Darshan H
authors.initials: DH
authors.lastname: Patel
authors.forename: Kwang Ho
authors.initials: KH
authors.lastname: Lee
authors.forename: Dae-Seok
authors.initials: DS
authors.lastname: Lee
authors.forename: Hyeun-Jong
authors.initials: HJ
authors.lastname: Bae
mesh_terms: D000595:Amino Acid Sequence; D001487:Basidiomycota / Q000201:enzymology* / Q000235:genetics*; D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D007218:Industrial Microbiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D016415:Sequence Alignment
chemical_list: D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase