Effect of introduced disulfide bond on thermal and methanol induced inactivation. (A) Structure of P. mirabilis lipase. Lid helices are shown in green, bound Ca2+ in blue, and catalytic triad as orange ball and stick. The location of the equivalent disulfide bond found in the B. cepacia lipase but absent in PML is labeled and shown as sticks. (B) Thermal inactivation as a function of temperature. Residual activity was measured after incubation for 1 hour. (C) Inactivation as a function of methanol concentration. Residual activity was measured after incubation for 2 hours after dilution to 10% methanol. (D) Time course of inactivation by 50% methanol. Samples were diluted to 10% methanol prior to assaying for residual activity. Results are the average of three independent experiments. Error-bars are omitted but are less than 5% in all cases. Lines are shown for clarity and represent the best-fit to the data points. WT PML(black circles), BCL (red open circles), and Dieselzyme 1 (blue squares).