title: Spore-displayed enzyme cascade with tunable stoichiometry.
abstract: Taking the advantages of inert and stable nature of endospores, we developed a biocatalysis platform for multiple enzyme immobilization on Bacillus subtilis spore surface. Among B. subtilis outer coat proteins, CotG mediated a high expression level of Clostridium thermocellum cohesin (CtCoh) with a functional display capability of ∼104molecules per spore of xylose reductase-C. thermocellum dockerin fusion protein (XR-CtDoc). By co-immobilization of phosphite dehydrogenase (PTDH) on spore surface via Ruminococcus flavefaciens cohesin-dockerin modules, regeneration of NADPH was achieved. Both xylose reductase (XR) and PTDH exhibited enhanced stability upon spore surface display. More importantly, by altering the copy numbers of CtCoh and RfCoh fused with CotG, the molar ratio between immobilized enzymes was adjusted in a controllable manner. Optimization of spore-displayed XR/PTDH stoichiometry resulted in increased yields of xylitol. In conclusion, endospore surface display presents a novel approach for enzyme cascade immobilization with improved stability and tunable stoichiometry. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:383-389, 2017.
authors.affiliation: Dept. of Chemical and Environmental Engineering, University of California, Riverside, CA, 92521.
authors.forename: Long
authors.initials: L
authors.lastname: Chen
authors.affiliation: Dept. of Chemical and Environmental Engineering, University of California, Riverside, CA, 92521.
authors.forename: Ashok
authors.initials: A
authors.lastname: Mulchandani
authors.affiliation: Dept. of Chemical and Environmental Engineering, University of California, Riverside, CA, 92521.
authors.forename: Xin
authors.identifier: 0000-0001-7491-7805
authors.initials: X
authors.lastname: Ge
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D002462:Cell Membrane / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D048013:Acetivibrio thermocellus / Q000201:enzymology / Q000235:genetics; D004798:Enzymes / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D013171:Spores, Bacterial / Q000201:enzymology* / Q000235:genetics*
chemical_list: D004798:Enzymes
keywords: cofactor regeneration; cohesin-dockerin interaction; enzyme cascade; enzyme immobilization; spore surface display