[en] A total of 59 bacteria samples from Antarctic sea water were collected and screened for their ability to produce alpha-amylase. The highest activity was recorded from an isolate identified as an Alteromonas species. The purified alpha-amylase shows a molecular mass of about 50,000 Da and a pI of 5.2. The enzyme is stable from pH 7.5 to 9 and has a maximal activity at pH 7.5. Compared with other alpha-amylases from mesophiles and thermophiles, the "cold enzyme" displays a higher activity at low temperature and a lower stability at high temperature. The psychrophilic alpha-amylase requires both Cl- and Ca2+ for its amylolytic activity. Br- is also quite efficient as an allosteric effector. The comparison of the amino acid composition with those of other alpha-amylases from various organisms shows that the cold alpha-amylase has the lowest content in Arg and Pro residues. This could be involved in the principle used by the psychrophilic enzyme to adapt its molecular structure to the low temperature of the environment.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Chessa, Jean-Pierre
Feller, Georges ; Université de Liège - ULiège > Département des sciences de la vie > Labo de biochimie
Gerday, Charles ; Université de Liège - ULiège > Services généraux (Faculté des sciences) > Relations académiques et scientifiques (Sciences)
Language :
English
Title :
Purification and Characterization of the Heat-Labile Alpha-Amylase Secreted by the Psychrophilic Bacterium Tac 240b
Aghajari, N., Feller, G., Gerday, C., and Haser, R. 1998. Crystal structures of the psychrophilic α-amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor. Protein Sci. 7: 567-572.
Arpigny, J.L., Feller, G., Davail, S., Génicot, S., Narinx, E., Zekhini, Z., and Gerday, C. 1994. Molecular adaptations of enzymes from thermophilic and psychrophilic organisms. Adv. Comp. Environ. Physiol. 20: 269-295.
Bernfeld, P. 1955. Amylases, α and β. Methods Enzymol. 1: 149-151.
Borders, C.L., Broadwater, J.A., Bekeny, P.A., Salmon, J.E., Lee, A.S., Eldridge, A.M., and Pett, V.B. 1994. A structural role for arginine in proteins: multiple hydrogen bonds to backbone carbonyl oxygen. Protein. Sci. 3: 541-548.
Dambmann, C., and Aunstrup, K. 1981. The variety of serine proteases and their industrial significance. In Proteinases and their inhibitors. Structure, function and applied aspects. Edited by V. Turk and L. Vitale. Pergamon Press, Oxford.
Davail, S., Feller, G., Narinx, E., and Gerday, C. 1994. Cold adaptations of proteins. J. Biol. Chem. 269: 17448-17453.
Feller, G., Lonhienne, T., Deroanne, C., Libioulle, C., Van Beeumen, F., and Gerday, C. 1992. Purification, characterization and nucleotide sequence of the thermolabile α-amylase from the Antarctic psychrotroph Alteromonas haloplanctis A 23. J. Biol. Chem. 267: 5217-5221.
Feller, G., Payan, F., Theys, M., Qian, R., Haser, R., and Gerday, C. 1994a. Stability and structural analysis of α-amylase from the antarctic psychrophile Alteromonas haloplanctis A 23. Eur. J. Biochem. 222: 441-447.
Feller, G., Narinx, E., Arpigny, J.L., Zekhini, Z., Swings, J., and Gerday, C. 1994b. Temperature dependence of growth, enzyme secretion and activity of psychrophilic Antarctic bacteria. Appl. Microbiol. Biotechnol. 41: 477-479.
Feller, G., Sonnet, P., and Gerday, C. 1995. The β-lactamase secreted by the Antarctic psychrophile Psychrobacter immobilis A8. Appl. Microbiol. Biotechnol. 61: 4474-4476.
Feller, G., le Bussy, O., Houssier, C., and Gerday, C. 1996. Structural and functional aspects of chloride binding to Alteromonas haloplanctis α-amylase. J. Biol. Chem. 271(39): 23836-23841.
Gounot, A.-M. 1991. Bacterial life at low temperature: physiological aspects and biotechnological implications. J. Appl. Bacteriol. 71: 386-397.
Hardy, F., Vriend, G., Veltman, O.R., van der Vinne, B., Veneme, G., and Eijsink, V.G.H. 1993. Stabilisation of Bacillus stearothermophilus neutral protease by introduction of prolines. FEBS Lett. 317: 898-892.
Herning, T., Yutani, K., Inaka, K., Kuroki, R., Matsushima, M., and Kikuchi, M. 1992. Role of proline residues in human lysozyme stability: a scanning calorimetric study combined with X-ray structure analysis of proline mutants. Biochemistry, 31: 7077-7085.
Hochachka, P.W., and Somero, G.N. 1984. Biochemical adaptations. Princeton University Press, Princeton, N.J.
Kobori, H., Sullivan, C.W., and Shizuya, H. 1984. Heat-labile phosphatase from Antarctic bacteria: rapid 5′ end-labelling of nucleic acid. Proc. Natl. Acad. Sci. U.S.A. 81: 6691-6695.
Levitzki, A., and Steer, M.L. 1974. The allosteric activation of mammalian α-amylases by chloride. Eur. J. Biochem. 41: 171-180.
Machius, M., Wiegand, G., and Huber, R. 1995. Crystal structure of calcium-depleted Bacillus licheniformis α-amylase at 2.2 Å resolution. J. Mol. Biol. 246: 545-559.
Markler, D.J., Farrington, G.K., and Wedler, F.C. 1981. Protein stability. Int. J. Pept. Protein Res. 18: 430-442.
Matthews, B.W., Nicholson, H., and Becktel, W.J. 1987. Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding. Proc. Natl. Acad. Sci. U.S.A. 84: 6663-6667.
Menendez-Arias, L., and Argos, P. 1989. Engineering protein thermal stability. Sequence statistics point to residue substitutions in α-helices. J. Mol. Biol. 206: 397-406.
Qian, M., Haser, R., and Payan, F. 1993. Structure and molecular model refinement of pig pancreatic α-amylase at 2.1 Å resolution. J. Mol. Biol. 231: 785-799.
Qian, M., Haser, R., Buisson, M., Duée, E., and Payan, F. 1994. The active center of a mammalian α-amylase. Structure of the complex of a pancreatic α-amylase with a carbohydrate inhibitor refined to 2.2 Å resolution. Biochemistry, 33(20): 6284-6294.
Raucher, E., Neumann, U., Scaich, E., Von Bulow, S., and Wahlefeld, A.M. 1985. Optimized conditions for determining activity concentration of α-amylase in serum, with 1,4-α-D-4-nitrophenylmaltoheptaoside as substrate. Clin. Chem. 31: 14-19.
Russel, N.J. 1990. Cold adaptation of microorganisms. Philos. Trans. R. Soc. London Ser. B, 326: 595-611.
Russel, N.J., and Fukunaga, N. 1990. A comparison of thermal adaptation of membrane lipids in psychrophilic and thermophilic bacteria. FEMS Microbiol. Rev. 75: 171-182.
Somero, G.N. 1977. Temperature as a selective factor in protein evolution: the adaptational strategy of « compromise ». J. Exp. Zool. 194: 175-188.
Suzuki, Y., Oishi, K., Nakano, H., and Nagayama, T. 1987. A strong correlation between the increase in number of proline residues and the rise in thermostability of five Bacillus oligo1,6-glucosidase. Appl. Microbiol. Biotechnol. 26: 546-551.
Svensson, B., and Sogaard, M. 1993. Mutational analysis of glycosylase function. J. Biotechnol. 29: 1-37.
Watanabe, K., Masuda, T., Ohashi, H., Mihara, H., and Suzuki Y. 1994. Multiple proline substitutions cumulatively thermostabilize Bacillus cereus ATCC7064 oligo-1,6-glucosidase. Eur. J. Biochem. 226: 277-283.