Ammonium sulfate precipitation and dialysis

All steps were performed at 4 °C unless otherwise stated. Fifty milliliters of CFS was used for precipitation with different concentrations of AS. Required quantity of solid AS was slowly added with concomitant mixing using a magnetic stirrer. The AS treated solutions were kept undisturbed for either 20 min (short term incubation; STI) or 3 h (long time incubation; LTI) or 8 h (extended time incubation; XTI). Protein pellet was collected by centrifugation at 4032xg for 30 min. The resultant protein pellets were dissolved in 800 μl of 50 mM potassium phosphate buffer (pH6.5) and either directly used for plate assay (laminarin and carboxy methyl cellulose (CMC)) or dialyzed for the reducing sugar assay. For the dialyzed samples, protein solutions containing AS were dialyzed against 50 mM potassium phosphate buffer (pH6.5) with three successive buffer changes over the period of 12 h using a 12 kDa cutoff membrane tubing. The dialyzed solutions were carefully collected, and their volumes measured.