Protein Engineering Protocols - Mycobacteriology research center

Directed Evolution of Thermostability 297Fig. 7. Enzyme kinetics and exponential decay of catalytic activity of N∆5-clone andstable kinetics of N∆5-S3/6 at 40°C. (A) Spectrophotometric analysis of product formationusing the chromogenic substrate nitrocefin. The final enzyme concentration was2.9 nM for N∆5-clone (¡ ) and 0.5 nM for N∆5-S3/6 clone (●). Twenty microliters ofa concentrated enzyme solution was mixed with 980 µL of prewarmed assay buffer.(B) Exponential decay fit of enzymatic activity of N∆5-clone using the change ofabsorbance per second (∆A/∆t) from the data shown in (A). A three-parameter exponentialdecay fit revealed a half-life of 7 s at 40°C (using SigmaPlot).1. Equilibrate 300 to 400 nM enzyme solutions from Subheading 3.8.3., step 4 containing0.25 to 8 M urea for 18 to 20 h at 19°C.2. Record fluorescence emission spectra from 320 to 380 nm at 20 to 23°C whileexciting at 280 nm. For each data point, average four scans.3. If necessary, correct fluorescence spectra for the background fluorescence of thesolution (phosphate buffer plus denaturant).