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

Abstract

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 Bacillus amyloliquifaciens (BAA) and Bacillus licheniformis (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.

Communicated by Ramaswamy H. Sarma

Keywords:

• Protein aggregation
• refolding
• osmolytes
• amino acids
• α-amylase

Acknowledgments

Authors are thankful to Prof. Rajiv Bhat, School of Biotechnology, Jawaharlal Nehru University for helpful discussion. The work is supported by a grant from UGC-MRP (MAJOR-BIOT-2013-32040) and DST-PURSE. Instrumentation facility provided by DBT-Builder (DBT-BUILDER, BT/PR5006/INF/153/2012) is gratefully acknowledged.

Author contributions

Aziz Ahmad: investigation, methodology, writing—original draft, writing—review and editing. Prachi Joshi: data analysis and writing—review and editing. Rajesh Mishra: conceptualization, funding acquisition, investigation, supervision, writing—review and editing.

Disclosure statement

No competing interest.

Data availability statement

Data will be made available on request.

Additional information

Funding

The work is supported by a grant from UGC (MAJOR-BIOT-2013-32040) and DST-PURSE.