Journal: Biomolecules

Article Title: Cold-Active Starch-Degrading Enzymes from a Cold and Alkaline Greenland Environment: Role of Ca 2+ Ions and Conformational Dynamics in Psychrophilicity

doi: 10.3390/biom15030415

Figure Lengend Snippet: Structure and stability of the three amylases, as measured by spectroscopy. ( a ) CD spectra of enzymes with/without 3 mM EDTA ( b ) Thermal stability of amylases in the presence or absence of 3 mM EDTA. Enzymes were cooled from room temperature to 10 °C and then heated at a scan rate of 1 °C/min, while full CD spectra were recorded (200–260 nm). Top: Selected traces monitored at different wavelengths in the α-helix region (214–230 nm) are shown (points), along with the unfolding fit (solid lines). ( c ) Example data of thermal unfolding, as measured by differential scanning fluorometry. Amylases were slowly heated from 15 to 95 °C at 1 °C/min (red squares and orange circles) and recooled to measure refolding (blue triangles), and the unfolding regime was fitted to a two-state unfolding (line). Red squares show the unfolding of Ika2 at 2 mM CaCl 2 , and orange circles show the unfolding of Ika2 with 2 mM CaCl 2 and 3mM EDTA. Data are the overlay of three technical repeats measured in parallel.

Article Snippet: Differential scanning fluorimetry: Loss of tertiary structure during heating was monitored with differential scanning fluorimetry on a NanoTemper Prometheus Panta with 7 μL enzyme (0.1 mg/mL) in 50 mM MOPS, 50 mM NaCl, 2 mM CaCl 2 .

Techniques: Spectroscopy, Circular Dichroism