title: Construction and characterization of chimeric cellulases with enhanced catalytic activity towards insoluble cellulosic substrates.
abstract: The chimeric proteins viz. CBM3-Cel9A, CBM4-Cel9A and CBM30-Cel9A, are constructed by fusion of family 3, 4, and 30 cellulose binding modules (CBMs) to N-terminus of family 9 endoglucanase (Cel9A) from Alicyclobacillus acidocaldrious. The chimeric enzymes were successfully expressed in Escherichia coli and purified to homogeneity. The chimeric enzymes showed significant increase in Avicel (8-12 folds) and filter paper (7-10 folds) degradation activities compared to Cel9A endoglucanase. Computational protein modeling and simulation on the chimeric enzymes were applied to analyze the fused CBMs effect on the increased insoluble cellulosic substrates degradation activity. Thin layer chromatography analysis of the enzymatic hydrolysis products and distribution of reducing sugars between soluble and insoluble fractions indicated processive cleavage of insoluble cellulosic substrates by the chimeras. The fused CBMs played a critical accessory role for the Cel9A catalytic domain and changed its character to facilitate the processive cleavage of insoluble cellulosic substrates.
authors.affiliation: Division of Applied Life Sciences (BK21), PMBBRC, Gyeongsang National University, 900 Gajwa-dong, Jinju, Gyeongnam 660-701, Republic of Korea.
authors.forename: Amar A
authors.initials: AA
authors.lastname: Telke
authors.forename: Sunil S
authors.initials: SS
authors.lastname: Ghatge
authors.forename: Seo-Hee
authors.initials: SH
authors.lastname: Kang
authors.forename: Sundarapandian
authors.initials: S
authors.lastname: Thangapandian
authors.forename: Keun-Woo
authors.initials: KW
authors.lastname: Lee
authors.forename: Hyun-Dong
authors.initials: HD
authors.lastname: Shin
authors.forename: Youngsoon
authors.initials: Y
authors.lastname: Um
authors.forename: Seon-Won
authors.initials: SW
authors.lastname: Kim
mesh_terms: D001419:Bacteria / Q000201:enzymology; D055162:Biocatalysis; D044602:Cellulases / Q000302:isolation & purification / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D002482:Cellulose; D044602:Cellulases