Stability of AsNAL

Long term stability of AsNAL at different pH values was studied by incubating the enzyme at pH 6.0 to 11.0 at room temperature for a month (S4A Fig). The decrease in activity was calculated compared to initial activity at the respective pH values. The enzyme activity in the condensation direction was measured using the standard reaction mixture incubated at room temperature for 1 h, with subsequent TBA assay. The pH stability was also studied using the thermofluor method [56]. For the thermofluor assay, the protein was dialyzed overnight at 4°C against a buffer containing 10 mM HEPES pH 7.5, 150 mM NaCl and 2 mM β-ME. The dialyzed protein was mixed with 2 μL of 300x Sypro Orange protein gel stain (Sigma-Aldrich, St. Louis, MO, USA) and 100 mM of different buffers ranging from pH 5.0 to pH 9.0 to a final volume of 25 μL (S4B Fig). Thermal shifts were screened for by heating in an iCycler iQ Real Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) from 1 to 80°C in increments of 1°C/min.

The melting temperature of AsNAL was studied by differential scanning calorimetry (DSC) using a Nano Differential Scanning Calorimeter III (Calorimetry Sciences Corporation, MA, USA). Protein was dialyzed overnight at 4°C against 50 mM HEPES pH 7.5 and 500 mM NaCl, filtered and then degassed for 15 min and concentrated to 1.9 mg/ml. Thermal denaturation was followed between 1 to 80°C using a heating/cooloing rate of 1°C/min and the dialysis buffer was used as reference buffer in the runs. The NanoAnalyze software was used to calculate the melting temperature by the substraction of the buffer-buffer baseline from the protein scan and fitting the data toa two-state transition model. The results are presented in the supporting information (S5 Fig).