Screening for thermal stability. Extant and ancestral CYP116B proteins were expressed in BL21(DE3) E. coli at 25 °C for 48 h and total P450 concentrations were determined in intact cells as described by Johnston et al. (2008). E. coli cell pellets were resuspended in whole cell spectral assay buffer (0.1 M potassium phosphate buffer pH 7.4, 20 mM D-glucose, 6 mM magnesium acetate) to a final concentration of 0.06 g wet cells per mL before incubation at a range of temperatures (25-80 °C) in a BioRad TM T100 thermal cycler (Bio-Rad Laboratories, Inc., Hercules, California, USA) for 15 or 60 min. The residual P450 content was then measured. The proportion of total P450 remaining was plotted against temperature and the 15 T50 or 60 T50 (i.e., the temperature at which 50 % of the P450 is lost after a 15-or 60-min incubation) was calculated using an IC50 curve fit in GraphPad Prism 8.0. Data are means of three replicates.