title: Molecular cloning and characterization of a β-glucanase from Piromyces rhizinflatus.
abstract: Cellulose is the most abundant renewable polysaccharide with a high potential for degradation to useful end products. In nature, most cellulose is produced as crystalline cellulose. Therefore, cellulases with high hydrolytic activity against crystalline cellulose are of great interest. In this study, a crystalline cellulose degradation enzyme was investigated. The cDNA encoding a β-glucanase, CbhYW23-2, was cloned from the ruminal fungus Piromyces rhizinflatus. To examine the enzyme activities, CbhYW23-2 was expressed in Escherichia coli as a recombinant His(6) fusion protein and purified by immobilized metal ion-affinity chromatography. Response surface modeling (RSM) combined with central composite design (CCD) and regression analysis was then employed for the planned statistical optimization of the β-glucanase activities of CbhYW23-2. The optimal conditions for the highest β-glucanase activity of CbhYW23-2 were observed at 46.4°C and pH 6.0. The results suggested that RSM combined with CCD and regression analysis were effective in determining optimized temperature and pH conditions for the enzyme activity of CbhYW23-2. CbhYW23-2 also showed hydrolytic activities toward Avicel, carboxymethyl cellulose (CMC), lichenan, and pachyman. The results also proved that the high activity of CbhYW23-2 on crystalline cellulose makes it a promising candidate enzyme for biotechnological and industrial applications.
authors.affiliation: Institute of Biotechnology, National Taiwan University, Taipei, Taiwan.
authors.forename: Chih-Yuan
authors.initials: CY
authors.lastname: Chu
authors.forename: Chih-Wen
authors.initials: CW
authors.lastname: Tseng
authors.forename: Pei-Ying
authors.initials: PY
authors.lastname: Yueh
authors.forename: Chung-Hang
authors.initials: CH
authors.lastname: Duan
authors.forename: Je-Ruei
authors.initials: JR
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D044602:Cellulases / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005936:Glucans / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D020089:Piromyces / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D005936:Glucans; D011994:Recombinant Proteins; D002482:Cellulose; D044602:Cellulases