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Fusion of a family 9 cellulose-binding module improves catalytic potential of Clostridium thermocellum cellodextrin phosphorylase on insoluble cellulose

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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).

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Acknowledgments

This work was not possible without support from the Biological Systems Engineering Department of Virginia Tech, the Air Force Office of Scientific Research (FA9550-08-1-0145), the USDA Biodesign and Bioprocess Center, and DOE BESC to YPZ.

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Authors and Affiliations

  1. Biological Systems Engineering Department, Virginia Tech, Blacksburg, VA, 24061, USA

    Xinhao Ye, Zhiguang Zhu, Chenming Zhang & Y.-H. Percival Zhang

  2. Institute for Critical Technology and Applied Science (ICTAS), Virginia Tech, Blacksburg, VA, 24061, USA

    Y.-H. Percival Zhang

  3. DOE Bioenergy Science Center, Oak Ridge, TN, 37831, USA

    Y.-H. Percival Zhang

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  1. Xinhao Ye
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  2. Zhiguang Zhu
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  4. Y.-H. Percival Zhang
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Correspondence to Y.-H. Percival Zhang.

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Ye, X., Zhu, Z., Zhang, C. et al. Fusion of a family 9 cellulose-binding module improves catalytic potential of Clostridium thermocellum cellodextrin phosphorylase on insoluble cellulose. Appl Microbiol Biotechnol 92, 551–560 (2011). https://doi.org/10.1007/s00253-011-3346-8

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