FIGURE 3.

Biochemical characterization of Calkro_0111. A, domain organization for full-length Calkro_0111 and TMs produced in E. coli used for characterization. B, optimum pH at 70 °C and optimum temperature at the optimum pH were determined for Calkro_0111 TM1 (0.9 mg/ml, incubated for 5 h) and TM8 (0.005 mg/ml, incubated for 30 min) on the substrate laminarin. Error bars, S.D. (n = 3). C, oligosaccharides released from laminarin by TM1 and TM8. HPLC chromatograms for each enzyme reaction time are aligned with respect to retention time and graphed together. The location of the peaks for standards glucose (G), laminaribiose (L2), laminaritriose (L3), laminaritetraose (L4), laminaripentaose (L5), and laminarihexaose (L6) are shown. D, truncation mutant activity as measured by the DNS reducing sugar assay. TM1 to TM5 and TM7 (4.2 μm) were incubated for 5 h, TM6 (12.3 μm) was incubated for 10 h, and TM8 to TM10 (0.2 μm) were incubated for 30 min with 1% (w/v) laminarin at 70 °C and pH 5 or 7. Activity is calculated in μmol of reducing sugar released/h/nmol of protein to compare activity for the proteins of different mass. Error bars, S.D. (n = 3).