Biochemical characterization of a pectate lyase from Caldicellulosiruptor bescii
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Caldicellulosiruptor bescii is a gram-positive thermophilic bacterium with the ability to grow on untreated plant biomass. Upon growth on switchgrass at 78°C, a set of genes encoding 94 proteins was up-regulated transcriptionally by more than 4-fold, in comparison to growth on glucose. Enzymes with activity towards pectin were among those that were most highly up-regulated. Of a total of four predicted pectinolytic enzymes, Cbes_1854, a multidomain protein of polysaccharide lyase family 3, was chosen for overexpression and purification in E. coli. The full-length protein (X-PL3) and just the catalytic domain (PL3) were purified with yields of approximately 10 mg/liter E. coli culture. Both purified recombinant proteins display alkaline pH optima and have a half-life of approximately one hour at 90°C. Under in vitro assay conditions, domain X does not play a significant role in catalysis. The catalytic domain, PL3, also shows a synergistic effect on sugar release from untreated switchgrass biomass during in vitro assays with a cellulase (Cel A) from C. bescii. In a collaborative study, the crystal structure of the PL3 domain was obtained. This provided insight into the catalytic mechanism of the enzyme.