title: Fusion of a family 9 cellulose-binding module improves catalytic potential of Clostridium thermocellum cellodextrin phosphorylase on insoluble cellulose.
abstract: Clostridium thermocellum cellodextrin phosphorylase (CtCDP), a single-module protein without an apparent carbohydrate-binding module, has reported activities on soluble cellodextrin with a degree of polymerization (DP) from two to five. In this study, CtCDP was first discovered to have weak activities on weakly water-soluble celloheptaose and insoluble regenerated amorphous cellulose (RAC). To enhance its activity on solid cellulosic materials, four cellulose binding modules, e.g., CBM3 (type A) from C. thermocellum CbhA, CBM4-2 (type B) from Rhodothermus marinus Xyn10A, CBM6 (type B) from Cellvibrio mixtus Cel5B, and CBM9-2 (type C) from Thermotoga maritima Xyn10A, were fused to the C terminus of CtCDP. Fusion of any selected CBM with CtCDP did not influence its kinetic parameters on cellobiose but affected the binding and catalytic properties on celloheptaose and RAC differently. Among them, addition of CBM9 to CtCDP resulted in a 2.7-fold increase of catalytic efficiency for degrading celloheptaose. CtCDP-CBM9 exhibited enhanced specific activities over 20% on the short-chain RAC (DP = 14) and more than 50% on the long-chain RAC (DP = 164). The chimeric protein CtCDP-CBM9 would be the first step to construct a cellulose phosphorylase for in vitro hydrogen production from cellulose by synthetic pathway biotransformation (SyPaB).
authors.affiliation: Biological Systems Engineering Department, Virginia Tech, Blacksburg, VA 24061, USA.
authors.forename: Xinhao
authors.initials: X
authors.lastname: Ye
authors.forename: Zhiguang
authors.initials: Z
authors.lastname: Zhu
authors.forename: Chenming
authors.initials: C
authors.lastname: Zhang
authors.forename: Y-H Percival
authors.initials: YH
authors.lastname: Zhang
mesh_terms: D002482:Cellulose / Q000378:metabolism*; D020633:Cellvibrio / Q000201:enzymology / Q000235:genetics; D048013:Acetivibrio thermocellus / Q000201:enzymology*; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D045245:Rhodothermus / Q000201:enzymology / Q000235:genetics; D020124:Thermotoga maritima / Q000201:enzymology / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D002482:Cellulose; D005964:Glucosyltransferases; C105024:cellodextrin phosphorylase