441824 Biomass Degradation By Multi-Domain Catalytic Surface-Layer Proteins in the Extremely Thermophilic Bacterium Caldicellulosiruptor Kronotskyensis

Monday, November 9, 2015
Exhibit Hall 1 (Salt Palace Convention Center)
George W. Harper, Jonathan M. Conway and Robert M. Kelly, Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC

Caldicellulosiruptor species are anaerobic thermophillic bacteria isolated from globally distributed terrestrial hot springs. There is interest in Caldicellulosiruptor species as model organisms for industrial biofuel processes for their large inventory of multi-domain biomass degradation enzymes for efficiently degrading cellulose and hemicellulose.  A subset of the Caldicellulosiruptor multi-domain enzymes have both catalytic glycoside hydrolase (GH) domains and surface layer homology (SLH) domains, which attach these enzymes to the surface of the cell.   We hypothesize that the localization of these enzymes at the interface between the cell and substrate improves the efficiency of biomass degradation by Caldicellulosiruptor species. Confocal immunofluorescence microscopy was performed on C. kronotskyensis using IgY polyclonal chicken antibodies raised against portions of two SLH GH enzymes, laminarinase Calkro_0111 and xylanase Calkro_0402, and confirmed that these enzymes are localized on the cell surface. To understand the biochemical function of these enzymes Calkro_0111 (2435 amino acids), the largest GH enzyme from the genus, was investigated. Two truncations of Calkro_0111 around each of the two catalytic domains (GH16 and GH55) were produced recombinantly in E. coli. The optimal temperature of 75°C and optimal pH of 5 for both of these enzyme truncations were determined on laminarin. In addition, HPLC analysis of the oligosaccharides released by these enzymes shows that the GH16 is an endo-glucanase, while the GH55 is an exo-glucanase.  Understanding the role of these large, multi-domain cell surface localized enzymes in biomass degradation will help to optimize enzymes in engineered strains to increase biofuel production.

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