title: Processivity and enzymatic mode of a glycoside hydrolase family 5 endoglucanase from Volvariella volvacea.
abstract: EG1 is a modular glycoside hydrolase family 5 endoglucanase from Volvariella volvacea consisting of an N-terminal carbohydrate-binding module (CBM1) and a catalytic domain (CD). The ratios of soluble to insoluble reducing sugar produced from filter paper after 8 and 24 h of exposure to EG1 were 6.66 and 8.56, respectively, suggesting that it is a processive endoglucanase. Three derivatives of EG1 containing a core domain only or additional CBMs were constructed in order to evaluate the contribution of the CBM to the processivity and enzymatic mode of EG1 under stationary and agitated conditions. All four enzymatic forms exhibited the same mode of action on both soluble and insoluble cellulosic substrates with cellobiose as a main end product. An additional CBM fused at either the N or C terminus reduced specific activity toward soluble and insoluble celluloses under stationary reaction conditions. Deletion of the CBM significantly decreased enzyme processivity. Insertion of an additional CBM also resulted in a dramatic decrease in processivity in enzyme-substrate reaction mixtures incubated for 0.5 h, but this effect was reversed when reactions were allowed to proceed for longer periods (24 h). Further significant differences were observed in the substrate adsorption/desorption patterns of EG1 and enzyme derivatives equipped with an additional CBM under agitated reaction conditions. An additional family 1 CBM improved EG1 processivity on insoluble cellulose under highly agitated conditions. Our data indicate a strong link between high adsorption levels and low desorption levels in the processivity of EG1 and possibly other processive endoglucanses.
authors.affiliation: Department of Biological Engineering, Nanjing Forestry University, Nanjing, Jiangsu, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Zheng
authors.forename: Shaojun
authors.initials: S
authors.lastname: Ding
mesh_terms: D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000032:analysis; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016254:Mutagenesis, Insertional; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D055367:Volvariella / Q000201:enzymology*
chemical_list: D002241:Carbohydrates; D050505:Mutant Proteins; D002480:Cellulase