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The noncellulosomal family 48 cellobiohydrolase from Clostridium phytofermentans ISDg: heterologous expression, characterization, and processivity

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Abstract

Family 48 glycoside hydrolases (cellobiohydrolases) are among the most important cellulase components for crystalline cellulose hydrolysis mediated by cellulolytic bacteria. Open reading frame (Cphy_3368) of Clostridium phytofermentans ISDg encodes a putative family 48 glycoside hydrolase (CpCel48) with a family 3 cellulose-binding module. CpCel48 was successfully expressed as two soluble intracellular forms with or without a C-terminal His-tag in Escherichia coli and as a secretory active form in Bacillus subtilis. It was found that calcium ion enhanced activity and thermostability of the enzyme. CpCel48 had high activities of 15.1 U μmol−1 on Avicel and 35.9 U μmol−1 on regenerated amorphous cellulose (RAC) with cellobiose as a main product and cellotriose and cellotetraose as by-products. By contrast, it had very weak activities on soluble cellulose derivatives (e.g., carboxymethyl cellulose (CMC)) and did not significantly decrease the viscosity of the CMC solution. Cellotetraose was the smallest oligosaccharide substrate for CpCel48. Since processivity is a key characteristic for cellobiohydrolases, the new initial false/right attack model was developed for estimation of processivity by considering the enzyme's substrate specificity, the crystalline structure of homologous Cel48 enzymes, and the configuration of cellulose chains. The processivities of CpCel48 on Avicel and RAC were estimated to be ∼3.5 and 6.0, respectively. Heterologous expression of secretory active cellobiohydrolase in B. subtilis is an important step for developing recombinant cellulolytic B. subtilis strains for low-cost production of advanced biofuels from cellulosic materials in a single step.

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Acknowledgments

This work was supported mainly by the DOE BioEnergy Science Center (BESC). The BioEnergy Science Center is a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. This work was also partially supported by the USDA Bioprocessing and Biodesign Center and DuPont Young Professor Award. Zuoming Zhang was partially supported by the China Scholar Council. Noppadon Sathitsuksanoh was partially supported by the ICTAS scholar program.

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Authors and Affiliations

  1. Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, 210-A Seitz Hall, Blacksburg, VA, 24061, USA

    Xiao-Zhou Zhang, Zuoming Zhang, Zhiguang Zhu, Noppadon Sathitsuksanoh & Y.-H. Percival Zhang

  2. Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, Changchun, 130023, China

    Zuoming Zhang

  3. Institute for Critical Technology and Applied Science (ICTAS), Virginia Polytechnic Institute and State University, Blacksburg, VA, 24061, USA

    Noppadon Sathitsuksanoh & Y.-H. Percival Zhang

  4. Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, 37831, USA

    Yunfeng Yang

  5. DOE BioEnergy Science Center (BESC), Oak Ridge, TN, 37831, USA

    Yunfeng Yang & Y.-H. Percival Zhang

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  1. Xiao-Zhou Zhang
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  2. Zuoming Zhang
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Correspondence to Y.-H. Percival Zhang.

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Zhang, XZ., Zhang, Z., Zhu, Z. et al. The noncellulosomal family 48 cellobiohydrolase from Clostridium phytofermentans ISDg: heterologous expression, characterization, and processivity. Appl Microbiol Biotechnol 86, 525–533 (2010). https://doi.org/10.1007/s00253-009-2231-1

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