Amino acids and glycine derivatives differently affect refolding of mesophilic and thermophilic like α-amylases: implications in protein refolding and aggregation.

α-amylases are industrially important enzymes which are used in different starch-based industries. They are adapted to different environmental conditions like extremes of temperature, pH and salinity. Herein, α-amylases from (BAA) and (BLA), representing mesophilic and thermophilic-like proteins, respectively, have been used to investigate the effect of naturally occurring osmolytes like arginine, proline, glycine and its methyl derivatives, sarcosine and betaine on their refolding. In this study, we have shown that among amino acids and glycine derivatives, betaine is the most promising osmolyte, while arginine and glycine exhibit moderately positive effect at their lower concentrations on the refolding of BAA only. Except betaine, all other osmolytes above 0.25 M showed inhibitory effect on the native enzyme activity of BLA and BAA. However, aggregation kinetics monitored by static light scattering indicates suppression of aggregation by all of these osmolytes. Further investigation by tryptophan and ANS fluorescence spectroscopy indicates the formation of compact hydrophobic core in the presence of the osmolytes. The morphology of protein aggregates having different sizes was visualized by atomic force microscopy ,and it was observed that amorphous aggregates of variable heights were formed. Our study highlights the importance of differential effects of arginine, proline, glycine, sarcosine and betaine on the native state as well as on refolding of BLA and BAA which may be helpful in devising strategies for developing effective protein formulation and prevention of aggregation of industrially and therapeutically important proteins.