In the present study an attempt was made to investigate the macromolecular crowding effect on functional attributes of α-amylase. High concentrations of sugar based cosolvents, (e.g., trehalose, sucrose, sorbitol, and glycerol) were used to mimic the macromolecular crowding environment (of cellular milieu) under in vitro conditions. To assess the effect of macromolecular crowding, the activity and structural properties of the enzyme were evaluated in the presence of different concentrations of the above cosolvents. Based on the results it is suggested that the macromolecular crowding significantly improves the catalytic efficiency of the enzyme with marginal change in the structure. Out of four cosolvents examined, trehalose was found to be the most effective in consistently enhancing thermal stability of the enzyme. Moreover, the relative effectiveness of the above cosolvents was found to be dependent on their concentration used. 1. Introduction Proteins are complex molecules and often unstable when they are placed out of their native environment. Proteins or enzymes may also lose their activity as a result of high temperature, aggregation, proteolysis, and suboptimal solution conditions. The purified proteins are often unstable and need to be stabilized in order to maintain the structural integrity and activity. Protein aggregation during processing and formulation is one of the major setbacks that limit the rapid commercialization of protein-based pharmaceuticals. Proteins aggregation is usually triggered by the formation of partially unfolded intermediates and therefore the demand for successful stabilization protocol is progressively increasing [1]. The phenomenon of stabilization of proteins infers the preservation of the native protein structure and activity during storage. The protein stabilization is based on the principle of limiting the molecular motion and conformational transition while maintaining the protein still in the native state [2, 3]. The native structure of a protein is dictated by various intramolecular interactions and its contacts with the surrounding solutes and solvents. Several sugars and polyols have extensively been used to stabilize the proteins against denaturation and to extend their shelf life during storage [4, 5]. The structural stability of a protein in an aqueous solution is determined by several types of weak interactions. The interactions between solvent, cosolvents, and protein have often been explained in terms of transfer free energy, preferential interaction parameters and preferential interaction
References
[1]
W. Wang, “Protein aggregation and its inhibition in biopharmaceutics,” International Journal of Pharmaceutics, vol. 289, no. 1-2, pp. 1–30, 2005.
[2]
L. L. Chang, D. Shepherd, J. Sun et al., “Mechanism of protein stabilization by sugars during freeze-drying and storage: native structure preservation, specific interaction, and/or immobilization in a glassy matrix?” Journal of Pharmaceutical Sciences, vol. 94, no. 7, pp. 1427–1444, 2005.
[3]
V. Ragoonanan and A. Aksan, “Protein stabilization,” Transfusion Medicine and Hemotherapy, vol. 34, no. 4, pp. 246–252, 2007.
[4]
Y. Fujita, Y. Iwasa, and Y. Noda, “Effect of polyhydric alcohols on invertase stabilization,” Bulletin of the Chemical Society of Japan, vol. 55, pp. 1896–1900, 1984.
[5]
K. Gekko, “Calorimetric study on thermal denaturation of lysozyme in polyol-water mixtures,” Journal of Biochemistry, vol. 91, no. 4, pp. 1197–1204, 1982.
[6]
T. Arakawa and S. N. Timasheff, “Preferential interactions of proteins with salts in concentrated solutions,” Biochemistry, vol. 21, no. 25, pp. 6545–6552, 1982.
[7]
S. N. Timasheff, “The control of protein stability and association by weak interactions with water: how do solvents affect these processes?” Annual Review of Biophysics and Biomolecular Structure, vol. 22, pp. 67–97, 1993.
[8]
M. B. Burg and J. D. Ferraris, “Intracellular organic osmolytes: function and regulation,” The Journal of Biological Chemistry, vol. 283, no. 12, pp. 7309–7313, 2008.
[9]
O. Bounedjah, L. Hamon, P. Savarin, B. Desforges, Curmi, and D. Pastre, “Macromolecular crowding regulates assembly of mRNA stress granules after osmotic stress,” The Journal of Biological Chemistry, vol. 287, pp. 2446–2458, 2012.
[10]
L. Hamon, P. Savarin, P. A. Curmi, and D. Pastre, “Rapid assembly and collective behavior of microtubule bundles in the presence of polyamines,” Biophysical Journal, vol. 101, pp. 205–216, 2011.
[11]
S. B. Zimmerman and S. O. Trach, “Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli,” Journal of Molecular Biology, vol. 222, no. 3, pp. 599–620, 1991.
[12]
G. K. Farber and G. A. Petsko, “The evolution of α/β barrel enzymes,” Trends in Biochemical Sciences, vol. 15, no. 6, pp. 228–234, 1990.
[13]
S. Janecek, “Alpha-amylase family: molecular biology and evolution,” Progress in Biophysics and Molecular Biology, vol. 67, pp. 67–97, 1997.
[14]
E. A. MacGregor, “α-Amylase structure and activity,” Journal of Protein Chemistry, vol. 7, no. 4, pp. 399–415, 1988.
[15]
L. Holm, A. K. Koivula, P. M. Lehtovaara, A. Hemminki, and J. K. C. Knowles, “Random mutagenesis used to probe the structure and function of Bacillus stearothermophilus alpha-amylase,” Protein Engineering, vol. 3, no. 3, pp. 181–191, 1990.
[16]
O. H. Lowry, N. J. Rosenbrough, A. L. Farr, and R. J. Randall, “Protein measurement with the Folin phenol reagent,” The Journal of Biological Chemistry, vol. 193, no. 1, pp. 265–275, 1951.
[17]
P. Bernfeld, “α- and β-Amylases,” Methods in Enzymology, vol. 1, pp. 149–158, 1955.
[18]
N. Declerck, M. Machius, P. Joyet, G. Wiegand, R. Huber, and C. Gaillardin, “Hyperthermostabilization of Bacillus licheniformisα-amylase and modulation of its stability over a 50°C temperature range,” Protein Engineering, vol. 16, no. 4, pp. 287–293, 2003.
[19]
J. C. Lee and S. N. Timasheff, “The stabilization of proteins by sucrose,” The Journal of Biological Chemistry, vol. 256, no. 14, pp. 7193–7201, 1981.
[20]
S. Rajendran, C. Radha, and V. Prakash, “Mechanism of solvent-induced thermal stabilization of α-amylase from Bacillus amyloliquefaciens,” International Journal of Peptide and Protein Research, vol. 45, no. 2, pp. 122–128, 1995.
[21]
S. N. Timasheff, “Control of protein stability and reactions by weakly interacting cosolvents: the simplicity of the complicated,” Advances in Protein Chemistry, vol. 51, pp. 355–432, 1998.
[22]
R. Santucci, F. Polizio, and A. Desideri, “Formation of a molten-globule-like state of cytochrome c induced by high concentrations of glycerol,” Biochimie, vol. 81, no. 7, pp. 745–750, 1999.
[23]
J. K. Yadav and V. Prakash, “Thermal stability of alpha-amylase in aqueous cosolvent systems,” Journal of Biosciences, vol. 34, no. 3, pp. 377–387, 2009.
[24]
S. N. Timasheff, “Protein-solvent preferential interactions, protein hydration, and the modulation of biochemical reactions by solvent components,” Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 15, pp. 9721–9726, 2002.
[25]
S. N. Timasheff, “Protein hydration, thermodynamic binding, and preferential hydration,” Biochemistry, vol. 41, no. 46, pp. 13473–13482, 2002.
