Ep gradient s thermocycler

To determine half‐inactivation temperatures (T5060), the residual esterase activity of the enzymes was determined 18. Purified enzyme solutions (10 μg·ml⁻¹) were incubated for 60 min at temperatures between 63 and 87 °C in an ep gradient S thermocycler (Eppendorf, Hamburg, Germany) in sodium borate buffer (50 mm, pH 8) with or without the addition of CaCl₂ (10 mm). The incubation was stopped by cooling the samples to 4 °C. The residual activity was determined in HEPES buffer (0.1 m, pH 8) with 0.5 mm pNPB. The enzymatic hydrolysis of pNPB was spectrophotometrically monitored for 5 min at 405 nm in a microplate reader (Biotek Power Wave XS, Winooski, VT, USA). All determinations were performed in triplicates. Half‐inactivation temperatures were calculated by nonlinear regression as described previously 18.

Reaction vials containing partially purified enzyme preparations (33.3 μg mL⁻¹) in 1 m potassium phosphate buffer (pH 8.0) were incubated at 65, 70 and 75 °C for 24 h. A control sample was stored at 4 °C. The residual enzyme activity was determined with polycaprolactone nanoparticles as substrate at 50 °C using a PowerWave XS plate reader at 600 nm (BioTek Instruments, Inc., Winooski, VT, USA). The PCL nanoparticles were prepared as described previously.^[48] An enzyme concentration of 5 μg mL⁻¹ in 1 m potassium phosphate buffer (pH 8.0) was used. Purified PHL7 (0.2 mg mL⁻¹) was incubated in 50 mm phosphate buffer between 65 °C and 74 °C for 4 h, 16 h and 24 h in an ep gradient S thermocycler (Eppendorf, Hamburg, Germany). The residual activity was determined with p‐nitrophenol butyrate (pNPB) as substrate at 25 °C in 100 mm phosphate buffer, pH 7.5 containing 0.5 mmpNPB and 10 % ethanol. The absorbance of p‐nitrophenol released was monitored at 405 nm. Measurements were performed with a Synergy Mx microplate reader (BioTek Instruments Inc., Winooski, VT, USA). Mean values ±S.D. for n=3 are shown.