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http://dx.doi.org/10.5483/BMBRep.2011.44.10.665

Temperature, organic solvent and pH stabilization of the neutral protease from Salinovibrio proteolyticus: significance of the structural calcium  

Asghari, S. Mohsen (Department of Biology, Faculty of Science, University of Guilan)
Khajeh, Khosro (Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University)
Dalfard, Arastoo Badoei (Department of Biology, Faculty of Science, Shahid Bahonar University of Kerman)
Pazhang, Mohammad (Department of Cellular and Molecular Biology, Faculty of Science, Azarbaijan University of Tarbiat Moallem)
Karbalaei-Heidari, Hamid Reza (Department of Biology, College of Sciences, Shiraz University)
Publication Information
BMB Reports / v.44, no.10, 2011 , pp. 665-668 More about this Journal
Abstract
In order to clarify the impact of Ca-binding sites (Ca1 and 2) on the conformational stability of neutral proteases (NPs), we have analyzed the thermal, pH and organic solvent stability of a NP variant, V189P/A195E/G203D/A268E (Q-mutant), from Salinovibrio proteolyticus. This mutant has shown to bind calcium more tightly than the wild-type (WT) at Ca1 and to possess Ca2. Q-mutant was resisted against autolysis, thermoinactivation and pH denaturation in a Ca-dependent manner and exhibited better activity in organic solvents compared to the WT enzyme. These results imply that Ca1 and Ca2 are important for the conformational stability of NPs.
Keywords
Autolysis; Neutral proteases; Organic solvent stability; pH stability; Thermostability;
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1 Badoei-Dalfard, A., Khajeh, K., Asghari, S. M., Ranjbar, B., and Karbalaei-Heidari, H. R. (2010) Enhanced activity and stability in the presence of organic solvents by increased active site polarity and stabilization of a surface loop in a metalloprotease. J. Biochem. 148, 231-238.   DOI   ScienceOn
2 Imanaka, T., Shibazaki, M., and Takagi, M. (1986) A new way of enhancing the thermostability of proteases. Nature 324, 695-697.   DOI   ScienceOn
3 Carrea, G., and Riva, S. (2000) Properties and synthetic applications of enzymes in organic solvents. Angew. Chem. Int. 39, 2226-2254.   DOI   ScienceOn
4 Fisher, C. L., and Pei, G. K. (1997) Modification of a PCR-based site-directed mutagenesis method. BioTechniques 23, 570-574.
5 Clapes, P., Torres-Luis, J., and Adlercreutz, P. (1995) Enzyme peptide synthesis in low water content systems: preparative enzymatic synthesis of (Leu)-and (Met)-enkephalin derivatives. Bioinorg. Ned. Chem. 3, 244-255.
6 Morihara, K. (1967) The specificity of various neutral and alkaline proteins from microorganisms. Biochem. Biophys. Res. Commun. 26, 657-661.
7 Eijsink, V. G. H., Veltman, O. R., Aukema, W., Vriend, G. and Venema, G. (1995) Structural determinants of the stability of thermolysin-like proteinases. Nat. Struct. Mol. Biol. 2, 374-379.   DOI   ScienceOn
8 Colmax, M., Jansonius, J. N., and Matthews, B. W. (1972) The structure of thermolysin: an electron density map at 2.3 Å resolution. J. Mol. Biol. 70, 701-724.   DOI
9 Pauptit, R. A., Karlsson, .R, Picot, D., Jenkins, J. A., Nikolaus-Reimer, A. S., and Jansonius, J. N. (1988) Crystal structure of neutral protease from Bacillus cereus refined at 3.0 ${\AA}$ resolution and comparison with the homologous but more thermostable enzyme thermolysin. J. Mol. Biol. 199, 525-537.   DOI
10 Thayer, P. M., Flaherty, K. M., and McKay, D. B. (1991) Three-dimensional structure of the elastase of Pseudomonas aeruginosa at 1.5-${\AA}$ resolution. J. Biol. Chem. 266, 2864-2871.
11 Veltman, O. R., Vriend, G., Van den Burg, B., Hardy, F., Venema, G., and Eijsink, V. G. H. (1997) Engineering thermolysin-like proteases whose thermostability is largely independent of calcium. FEBS Lett. 405, 241-244.   DOI   ScienceOn
12 Asghari, S. M., Pazhang, M., Ehtesham, S., Karbalaei-Heidari, H. R., Taghdir, M., Sadeghizadeh, M., Naderi-Manesh, H. and Khajeh, K. (2010) Remarkable improvements of a neutral protease activity and stability share the same structural origins. Protein Eng. Des. Sel. 23, 599-606.   DOI   ScienceOn