Nε-Acetyl L-α Lysine Improves Activity and Stability of α-Amylase at Acidic Conditions: A Comparative Study with other Osmolytes

Author(s): Nidhya N. Joghee, Gurunathan Jayaraman*, Masilamani Selladurai

Journal Name: Protein & Peptide Letters

Volume 27 , Issue 6 , 2020

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Graphical Abstract:


Background: Nε-acetyl L-α lysine is an unusual acetylated di-amino acid synthesized and accumulated by certain halophiles under osmotic stress. Osmolytes are generally known to protect proteins and other cellular components under various stress conditions.

Objective: The structural and functional stability imparted by Nε-acetyl L-lysine on proteins were unknown and hence was studied and compared to other commonly known bacterial osmolytes - ectoine, proline, glycine betaine, trehalose and sucrose.

Methods: Effects of osmolytes on the temperature and pH profiles, pH stability and thermodynamic stability of the model enzyme, α-amylase were analyzed.

Results: At physiological pH, all the osmolytes under study increased the optimal temperature for enzyme activity and improved the thermodynamic stability of the enzyme. At acidic conditions (pH 3.0), Nε-acetyl L-α lysine and ectoine improved both the catalytic and thermodynamic stability of the enzyme; it was reflected in the increase in residual enzyme activity after incubation of the enzyme at pH 3.0 for 15 min by 60% and 63.5% and the midpoint temperature of unfolding transition by 11°C and 10°C respectively.

Conclusion: Such significant protective effects on both activity and stability of α-amylase imparted by addition of Nε-acetyl L-α lysine and ectoine at acidic conditions make these osmolytes interesting candidates for biotechnological applications.

Keywords: Osmolytes, alpha-amylase, enzyme activity, thermodynamic stability, Nε-acetyl-L-lysine, ectoine.

Oren, A. Diversity of halophilic microorganisms: environments, phylogeny, physiology, and applications. J. Ind. Microbiol. Biotechnol., 2002, 28(1), 56-63.
[http://dx.doi.org/10.1038/sj/jim/7000176] [PMID: 11938472]
Ventosa, A. Unusual micro-organisms from unusual habitats: Hypersaline environments, Symposia-society for general microbiology; Cambridge University Press: Cambridge, 2006, p. 223.
Bolen, D.W.; Baskakov, I.V. The osmophobic effect: natural selection of a thermodynamic force in protein folding. J. Mol. Biol., 2001, 310(5), 955-963.
[http://dx.doi.org/10.1006/jmbi.2001.4819] [PMID: 11502004]
Kim, Y.S.; Jones, L.S.; Dong, A.; Kendrick, B.S.; Chang, B.S.; Manning, M.C.; Randolph, T.W.; Carpenter, J.F. Effects of sucrose on conformational equilibria and fluctuations within the native-state ensemble of proteins. Protein Sci., 2003, 12(6), 1252-1261.
[http://dx.doi.org/10.1110/ps.0242603] [PMID: 12761396]
Timasheff, S.N. Protein hydration, thermodynamic binding, and preferential hydration. Biochemistry, 2002, 41(46), 13473-13482.
[http://dx.doi.org/10.1021/bi020316e] [PMID: 12427007]
Bourot, S.; Sire, O.; Trautwetter, A.; Touzé, T.; Wu, L.F.; Blanco, C.; Bernard, T. Glycine betaine-assisted protein folding in a lysA mutant of Escherichia coli. J. Biol. Chem., 2000, 275(2), 1050-1056.
[http://dx.doi.org/10.1074/jbc.275.2.1050] [PMID: 10625645]
Diamant, S.; Rosenthal, D.; Azem, A.; Eliahu, N.; Ben-Zvi, A.P.; Goloubinoff, P. Dicarboxylic amino acids and glycine-betaine regulate chaperone-mediated protein-disaggregation under stress. Mol. Microbiol., 2003, 49(2), 401-410.
[http://dx.doi.org/10.1046/j.1365-2958.2003.03553.x] [PMID: 12828638]
Fisher, M.T. Proline to the rescue. Proc. Natl. Acad. Sci. USA, 2006, 103(36), 13265-13266.
[http://dx.doi.org/10.1073/pnas.0606106103] [PMID: 16938858]
Ignatova, Z.; Gierasch, L.M. Inhibition of protein aggregation in vitro and in vivo by a natural osmoprotectant. Proc. Natl. Acad. Sci. USA, 2006, 103(36), 13357-13361.
[http://dx.doi.org/10.1073/pnas.0603772103] [PMID: 16899544]
Brown, A.D. Microbial water stress., 1976.
Santos, H.; da Costa, M.S. Compatible solutes of organisms that live in hot saline environments. Environ. Microbiol., 2002, 4(9), 501-509.
[http://dx.doi.org/10.1046/j.1462-2920.2002.00335.x] [PMID: 12220406]
Welsh, D.T. Ecological significance of compatible solute accumulation by micro-organisms: from single cells to global climate. FEMS Microbiol. Rev., 2000, 24(3), 263-290.
[http://dx.doi.org/10.1111/j.1574-6976.2000.tb00542.x] [PMID: 10841973]
Margesin, R.; Schinner, F. Potential of halotolerant and halophilic microorganisms for biotechnology. Extremophiles, 2001, 5(2), 73-83.
[http://dx.doi.org/10.1007/s007920100184] [PMID: 11354458]
Lentzen, G.; Schwarz, T. Extremolytes: Natural compounds from extremophiles for versatile applications. Appl. Microbiol. Biotechnol., 2006, 72(4), 623-634.
[http://dx.doi.org/10.1007/s00253-006-0553-9] [PMID: 16957893]
Chen, T.H.; Murata, N. Enhancement of tolerance of abiotic stress by metabolic engineering of betaines and other compatible solutes. Curr. Opin. Plant Biol., 2002, 5(3), 250-257.
[http://dx.doi.org/10.1016/S1369-5266(02)00255-8] [PMID: 11960744]
Nakayama, H.; Yoshida, K.; Ono, H.; Murooka, Y.; Shinmyo, A. Ectoine, the compatible solute of Halomonas elongata, confers hyperosmotic tolerance in cultured tobacco cells. Plant Physiol., 2000, 122(4), 1239-1247.
[http://dx.doi.org/10.1104/pp.122.4.1239] [PMID: 10759521]
Rodríguez-Salazar, J.; Suárez, R.; Caballero-Mellado, J.; Iturriaga, G. Trehalose accumulation in Azospirillum brasilense improves drought tolerance and biomass in maize plants. FEMS Microbiol. Lett., 2009, 296(1), 52-59.
[http://dx.doi.org/10.1111/j.1574-6968.2009.01614.x] [PMID: 19459961]
Moghaieb, R.E.; Abdel-Hadi, A-H.A.; Talaat, N.B. Molecular markers associated with salt tolerance in Egyptian wheats. Afr. J. Biotechnol., 2011, 10(79), 18092-18103.
Pastor, J.M.; Salvador, M.; Argandoña, M.; Bernal, V.; Reina-Bueno, M.; Csonka, L.N.; Iborra, J.L.; Vargas, C.; Nieto, J.J.; Cánovas, M. Ectoines in cell stress protection: uses and biotechnological production. Biotechnol. Adv., 2010, 28(6), 782-801.
[http://dx.doi.org/10.1016/j.biotechadv.2010.06.005] [PMID: 20600783]
Arakawa, T.; Ejima, D.; Kita, Y.; Tsumoto, K. Small molecule pharmacological chaperones: From thermodynamic stabilization to pharmaceutical drugs. Biochim. Biophys. Acta, 2006, 1764(11), 1677-1687.
[http://dx.doi.org/10.1016/j.bbapap.2006.08.012] [PMID: 17046342]
Rabbani, G.; Choi, I. Roles of osmolytes in protein folding and aggregation in cells and their biotechnological applications. Int. J. Biol. Macromol., 2018, 109, 483-491.
[http://dx.doi.org/10.1016/j.ijbiomac.2017.12.100] [PMID: 29274422]
Street, T.O.; Bolen, D.W.; Rose, G.D. A molecular mechanism for osmolyte-induced protein stability. Proc. Natl. Acad. Sci. USA, 2006, 103(38), 13997-14002.
[http://dx.doi.org/10.1073/pnas.0606236103] [PMID: 16968772]
Diamant, S.; Eliahu, N.; Rosenthal, D.; Goloubinoff, P. Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses. J. Biol. Chem., 2001, 276(43), 39586-39591.
[http://dx.doi.org/10.1074/jbc.M103081200] [PMID: 11517217]
Shimizu, S.; Smith, D.J. Preferential hydration and the exclusion of cosolvents from protein surfaces. J. Chem. Phys., 2004, 121(2), 1148-1154.
[http://dx.doi.org/10.1063/1.1759615] [PMID: 15260652]
Galinski, E.A.; Stein, M.; Amendt, B.; Kinder, M. The kosmotropic (structure-forming) effect of compensatory solutes. Comp. Biochem. Physiol. Part A. Physiol., 1997, 117(3), 357-365.
Da Costa, M.S.; Santos, H.; Galinski, E.A. An overview of the role and diversity of compatible solutes in bacteria and archaea. Adv. Biochem. Eng. Biotechnol., 1998, PP, 117-153.
Wohlfarth, A.; Severin, J.; Galinski, E.A. Identification of N δ-acetylornithine as a novel osmolyte in some Gram-positive halophilic eubacteria. Appl. Microbiol. Biotechnol., 1993, 39(4-5), 568-573.
Del Moral, A.; Severin, J.; Ramos-Cormenzana, A.; Trüper, H.G.; Galinski, E.A. Compatible solutes in new moderately halophilic isolates. FEMS Microbiol. Lett., 1994, 122(1-2), 165-172.
Saum, S.H.; Pfeiffer, F.; Palm, P.; Rampp, M.; Schuster, S.C.; Müller, V.; Oesterhelt, D. Chloride and organic osmolytes: a hybrid strategy to cope with elevated salinities by the moderately halophilic, chloride-dependent bacterium Halobacillus halophilus. Environ. Microbiol., 2013, 15(5), 1619-1633.
[http://dx.doi.org/10.1111/j.1462-2920.2012.02770.x] [PMID: 22583374]
Joghee, N.N.; Jayaraman, G. Metabolomic characterization of halophilic bacterial isolates reveals strains synthesizing rare diaminoacids under salt stress. Biochimie, 2014, 102, 102-111.
[http://dx.doi.org/10.1016/j.biochi.2014.02.015] [PMID: 24636996]
Xiao, Z.; Storms, R.; Tsang, A. A quantitative starch-iodine method for measuring alpha-amylase and glucoamylase activities. Anal. Biochem., 2006, 351(1), 146-148.
[http://dx.doi.org/10.1016/j.ab.2006.01.036] [PMID: 16500607]
Knapp, S.; Ladenstein, R.; Galinski, E.A. Extrinsic protein stabilization by the naturally occurring osmolytes β-hydroxyectoine and betaine. Extremophiles, 1999, 3(3), 191-198.
[http://dx.doi.org/10.1007/s007920050116] [PMID: 10484175]
Kaushik, J.K.; Bhat, R. Why is trehalose an exceptional protein stabilizer? An analysis of the thermal stability of proteins in the presence of the compatible osmolyte trehalose. J. Biol. Chem., 2003, 278(29), 26458-26465.
[http://dx.doi.org/10.1074/jbc.M300815200] [PMID: 12702728]
Singh, L.R.; Dar, T.A.; Rahman, S.; Jamal, S.; Ahmad, F. Glycine betaine may have opposite effects on protein stability at high and low pH values. Biochim. Biophys. Acta, 2009, 1794(6), 929-935.
[http://dx.doi.org/10.1016/j.bbapap.2009.02.005] [PMID: 19254782]
Jamal, S.; Poddar, N.K.; Singh, L.R.; Dar, T.A.; Rishi, V.; Ahmad, F. Relationship between functional activity and protein stability in the presence of all classes of stabilizing osmolytes. FEBS J., 2009, 276(20), 6024-6032.
[http://dx.doi.org/10.1111/j.1742-4658.2009.07317.x] [PMID: 19765077]
Yadav, J.K.; Prakash, V. Thermal stability of α-amylase in aqueous cosolvent systems. J. Biosci., 2009, 34(3), 377-387.
[http://dx.doi.org/10.1007/s12038-009-0044-0] [PMID: 19805899]
Samuel, D.; Kumar, T.K.S.; Ganesh, G.; Jayaraman, G.; Yang, P-W.; Chang, M-M.; Trivedi, V.D.; Wang, S-L.; Hwang, K-C.; Chang, D-K.; Yu, C. Proline inhibits aggregation during protein refolding. Protein Sci., 2000, 9(2), 344-352.
[http://dx.doi.org/10.1110/ps.9.2.344] [PMID: 10716186]
Xia, Y.; Park, Y-D.; Mu, H.; Zhou, H-M.; Wang, X-Y.; Meng, F-G. The protective effects of osmolytes on arginine kinase unfolding and aggregation. Int. J. Biol. Macromol., 2007, 40(5), 437-443.
[http://dx.doi.org/10.1016/j.ijbiomac.2006.10.004] [PMID: 17173966]
James, S.; McManus, J.J. Thermal and solution stability of lysozyme in the presence of sucrose, glucose, and trehalose. J. Phys. Chem. B, 2012, 116(34), 10182-10188.
[http://dx.doi.org/10.1021/jp303898g] [PMID: 22909409]
Ou, W-B.; Park, Y-D.; Zhou, H-M. Effect of osmolytes as folding aids on creatine kinase refolding pathway. Int. J. Biochem. Cell Biol., 2002, 34(2), 136-147.
[http://dx.doi.org/10.1016/S1357-2725(01)00113-3] [PMID: 11809416]
Van-Thuoc, D.; Guzmán, H.; Quillaguamán, J.; Hatti-Kaul, R. High productivity of ectoines by Halomonas boliviensis using a combined two-step fed-batch culture and milking process. J. Biotechnol., 2010, 147(1), 46-51.
[http://dx.doi.org/10.1016/j.jbiotec.2010.03.003] [PMID: 20223266]

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Year: 2020
Page: [551 - 556]
Pages: 6
DOI: 10.2174/0929866526666191105130041
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