Abstract
The halotolerance of a cold adapted α-amylase from the psychrophilic bacterium Pseudoalteromonas haloplanktis (AHA) was investigated. AHA exhibited hydrolytic activity over a broad range of NaCl concentrations (0.01–4.5 M). AHA showed 28% increased activity in 0.5–2.0 M NaCl compared to that in 0.01 M NaCl. In contrast, the corresponding mesophilic (Bacillus amyloliquefaciens) and thermostable (B. licheniformis) α-amylases showed a 39 and 46% decrease in activity respectively. Even at 4.5 M NaCl, 80% of the initial activity was detected for AHA, whereas the mesophilic and thermostable enzymes were inactive. Besides an unaltered fluorescence emission and secondary structure, a 10°C positive shift in the temperature optimum, a stabilization factor of >5 for thermal inactivation and a ΔT m of 8.3°C for the secondary structure melting were estimated in 2.7 M NaCl. The higher activation energy, half-life time and T m indicated reduced conformational dynamics and increased rigidity in the presence of higher NaCl concentrations. A comparison with the sequences of other halophilic α-amylases revealed that AHA also contains higher proportion of small hydrophobic residues and acidic residues resulting in a higher negative surface potential. Thus, with some compromise in cold activity, psychrophilic adaptation has also manifested halotolerance to AHA that is comparable to the halophilic enzymes.
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Abbreviations
- AHA:
-
Pseudoalteromonas haloplanktis α-amylase
- PPA:
-
Porcine pancreatic α-amylase
- BAA:
-
Bacillus amyloliquefaciens α-amylase
- BLA:
-
Bacillus licheniformis α-amylase
References
Aghajari N, Feller G, Gerday C, Haser R (1998) Structures of the psychrophilic Alteromonas haloplanctis α-amylase give insights into cold adaptation at a molecular level. Structure 6:1503–1516
Aghajari N, Feller G, Gerday C, Haser R (2002) Structural basis of α-amylase activation by chloride. Protein Sci 11:1435–1441
Coronado M, Vargas C, Hofemeister J, Ventosa A, Nieto JJ (2000) Production and biochemical characterization of an α-amylase from the moderate halophile Halomonas meridiana. FEMS Microbiol Lett 183:67–71
D’Amico S, Gerday C, Feller G (2001) Structural determinants of cold adaptation and stability in a large protein. J Biol Chem 276:25791–25796
D’Amico S, Marx J-C, Gerday C, Feller G (2003) Activity-stability relationships in extremophilic enzymes. J Biol Chem 278:7891–7896
Danson MJ, Hough DW (1997) The structural basis of protein halophilicity. Comp Biochem Physiol 117A:307–312
Dym O, Mevarech M, Sussman JL (1994) Structural features that stabilize halophilic malate dehydrogenase from an archaebacterium. Science 267:1344–1346
Feller G (2003) Molecular adaptations to cold in psychrophilic enzymes. Cell Mol Life Sci 60:648–662
Feller G, D’Amico S, Gerday C (1999) Thermodynamic stability of a cold-active α-amylase from the Antarctic bacterium Alteromonas haloplanktis. Biochemistry 38:4613–4619
Feller G, Lonhienne T, Deroanne C, Libioulle C, Beeumen JV, Gerday C (1992) Purification, characterization, and nucleotide sequence of the thermolabile α-amylase from the Antarctic psychrotroph Alteromonas haloplanctis A23. J Biol Chem 267:5217–5221
Georlette D, Blaise V, Collins T, D’Amico S, Gratia E, Hoyoux A, Marx J-C, Sonan G, Feller G, Gerday C (2004) Some like it cold: biocatalysis at low temperatures. FEMS Microbiol Rev 28:25–42
Gerday C, Aittaleb M, Bentahir M, Chessa JP, Claverie P, Collins T, D’Amico S, Dumont J, Garsoux G, Georlette D, Hoyoux A, Lonhienne T, Meuwis MA, Feller G (2000) Cold-adapted enzymes: from fundamentals to biotechnology. Trends Biotechnol 18:103–107
Hutcheon GW, Vasisht N, Bolhuis A (2005) Characterisation of a highly stable α-amylase from the halophilic archaeon Haloarcula hispanica. Extremophiles 6:487–495
Ishibashi M, Arakawa T, Philo JS, Sakashita K, Yonezawa Y, Tokunaga H, Tokunaga M (2002) Secondary and quaternary structural transition of the halophilic archaeon nucleotide diphosphate kinase under high- and low-salt conditions. FEMS Microbiol Lett 216:235–241
Kobayashi T, Kanai H, Aono R, Horikoshi K, Kudo T (1994) Cloning, expression, and nucleotide sequence of the α-amylase gene from the haloalkaliphilic archaeon Natronococcus sp strain Ah-36. J Bacteriol 176:5131–5134
Lowry OH, Rosebrough NJ, Farr AL, Randall R (1951) Protein measurement with Folin phenol reagent. J Biol Chem 193:265–275
Machius M, Declerck N, Huber R, Wiegand G (1998) Activation of Bacillus licheniformis α-amylase through a disorder→order transition of the substrate-binding site mediated by a calcium–sodium–calcium metal triad. Structure 6:281–292
Madern D, Camacho M, Rodriguez-Arnedo A, Bonete MJ, Zaccai G (2004) Salt-dependent studies of NADP-dependent isocitrate dehydrogenase from the halophilic archaeon Haloferax volcanii. Extremophiles 8:377–384
Madern D, Ebel C, Zaccai G (2000) Halophilic adaptation of enzyme. Extremophiles 4:91–98
Madern D, Zaccai G (2004) Molecular adaptation: the malate dehydrogenase from the extreme halophilic bacterium Salinibacter ruber behaves like a non-halophilic protein. Biochimie 86:295–303
Madigan MT, Marrs BL (1997) Extremophiles. Sci Am 276:82–87
Mevarech M, Frolow F, Gloss LM (2000) Halophilic enzymes: proteins with a grain of salt. Biophys Chem 86:155–164
Perez-Pomares F, Bautista V, Ferrer J, Pire C, Marhuenda-Egea FC, Bonete MJ (2003) α-Amylase activity from the halophilic archaeon Haloferax mediterranei. Extremophiles 7:299–306
Pflüger K, Müller V (2004) Transport of compatible solutes in extremophiles. J Bioenerg Biomembr 36:17–24
Polosina YY, Zamyatkin DF, Kostyukova AS, Filimonov VV, Fedorov OV (2002) Stability of Natrialba magadii NDPKinase: comparisons with other halophilic proteins. Extremophiles 6:135–142
Premkumar L, Greenblatt HM, Bageshwar UK, Savchenko T, Gokhman I, Sussman JL, Zamir A (2005) Three-dimensional structure of a halotolerant algal carbonic anhydrase predicts halotolerance of a mammalian homolog. Proc Natl Acad Sci USA 102:7493–7498
Record MTJ, Zhang W, Anderson CF (1998) Analysis of effects of salts and uncharged solutes on protein and nucleic acid equilibria and processes: a practical guide to recognizing and interpreting polyelectric effects, Hofmeister effects and osmotic effects of salts. Adv Protein Chem 51:281–353
Siddiqui KS, Poljak A, Guilhaus M, Feller G, D’Amico S, Gerday C, Cavicchioli R (2005) Role of disulfide bridges in the activity and stability of a cold-active α-amylase. J Bacteriol 187:6206–6212
Sivakumar N, Li N, Tang JW, Patel BK, Swaminathan K (2006) Crystal structure of AmyA lacks acidic surface and provide insights into protein stability at poly-extreme condition. FEBS Lett 580:2646–2652
Smalås AO, Heimstad ES, Hordvik A, Willassen NP, Male R (1994) Cold adaptation of enzymes: structural comparison between salmon and bovine trypsins. Proteins 20:149–166
Srimathi S, Jayaraman G (2005) Effect of glycosylation on the catalytic and conformational stability of homologous α-amylases. Protein J 24:79–88
Sundaram PV, Srimathi S (2004) Analysis of catalytic and structural stability of native and covalently modified enzymes. In: Svendsen A (ed) Enzyme functionality: design, engineering and screening. Marcel Dekker, New York, pp 632–661
Timasheff SN (1998) Control of protein stability and reactions by weakly interacting cosolvents: the simplicity of the complicated. Adv Protein Chem 51:355–432
Venkatesh R, Srimathi S, Yamuna A, Jayaraman G (2005) Enhanced catalytic and conformational stability of Atlantic cod trypsin upon neoglycosylation. Biochim Biophys Acta 1722:113–115
Wright DB, Banks DD, Lohman JR, Hilsenbeck JL, Gloss LM (2002) The effect of salts on the activity and stability of Escherichia coli and Haloferax volcanii dihydrofolate reductases. J Mol Biol 323:327–344
Acknowledgments
The work was supported by the Department of Biotechnology, Ministry of Science and Technology, Government of India. SS was partly supported by Swedish Institute Scholarship. We acknowledge Dr. Michael Danson, Dr. Sussan Crenell and Sridhar for the valuable inputs.
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Communicated by F. Robb.
This article is dedicated to Late Dr. P. V. Sundaram.
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Srimathi, S., Jayaraman, G., Feller, G. et al. Intrinsic halotolerance of the psychrophilic α-amylase from Pseudoalteromonas haloplanktis . Extremophiles 11, 505–515 (2007). https://doi.org/10.1007/s00792-007-0062-5
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DOI: https://doi.org/10.1007/s00792-007-0062-5