1887

Abstract

A recombinant clone expressing an amylase was identified from an generated genomic library of the thermophilic, moderately halophilic, anaerobic bacterium by activity screening, and the gene encoding the enzyme was designated AmyA. The gene was 1545 bp long, and encoded a 515 residue protein composed of a 25 amino acid putative signal peptide and a 490 amino acid mature protein. It possessed the five consensus regions characteristic of the α-amylase family and showed the greatest homology to the group of α-amylases. The gene was expressed in as a hexahistidine-tagged enzyme and purified. The purified recombinant enzyme was optimally active at 65 °C in 5% (w/v) NaCl at pH 75, with significant activity retained in the presence of up to 25% (w/v) NaCl. It had a specific activity of 2232 U mg and required NaCl and CaCl for optimum activity and thermostability. The relatively high proportion of acidic amino acids typically observed for many enzymes from halophiles was absent in AmyA.

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2002-08-01
2024-03-28
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References

  1. Abe J., Shibata Y., Fujisue M., Hizukuri S. 1996; Expression of periplasmic α-amylase of Xanthomonas campestris K-11151 in Escherichia coli and its action on maltose. Microbiology 142:1505–1512 [CrossRef]
    [Google Scholar]
  2. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. 1990; Basic local alignment search tool. J Mol Biol 215:403–410 [CrossRef]
    [Google Scholar]
  3. Benson D. A., Karsch-Mizrachi I., Lipman D. J., Ostell J., Rapp B. A., Wheeler D. L. 2000; GenBank. Nucleic Acids Res 28:15–18 [CrossRef]
    [Google Scholar]
  4. Besemer J., Borodovsky M. 1999; Heuristic approach to deriving models for gene finding. Nucleic Acids Res 27:3911–3920 [CrossRef]
    [Google Scholar]
  5. Brumm P. J., Hebeda R. E., Teague W. M. 1996; Purification and characterisation of the commercialized, cloned Bacillus megaterium α-amylase. Part II: transferase properties. Starch/Sterke 43:319–323
    [Google Scholar]
  6. Cayol J.-L., Ollivier B., Patel B. K. C., Prensier G., Guezennec J., Garcia J.-L. 1994; Isolation and characterization of Halothermothrix orenii gen. nov., sp. nov., a halophilic, thermophilic, fermentative, strictly anaerobic bacterium. Int J Syst Bacteriol 44:534–540 [CrossRef]
    [Google Scholar]
  7. Cayol J. L., Ducerf S., Patel B. K. C., Garcia J.-L., Thomas P., Ollivier B. 2000; Thermohalobacter berriensis gen. nov., sp. nov, a thermophilic, strictly halophilic bacterium from a solar saltern. Int J Syst Evol Microbiol 50:559–564 [CrossRef]
    [Google Scholar]
  8. Coronado M., Vargas C., Hofemeister J., Ventosa A., Nieto J. J. 2000a; Production and biochemical characterization of an α-amylase from the moderate halophile Halomonas meridiana . FEMS Microbiol Lett 183:67–71
    [Google Scholar]
  9. Coronado M. J., Vargas C., Mellado E., Tegos G., Drainas C., Nieto J. J., Ventosa A. 2000b; The α-amylase gene amyH of the moderate halophile Halomonas meridiana : cloning and molecular characterization. Microbiology 146:861–868
    [Google Scholar]
  10. Good W. A., Hartman P. A. 1970; Properties of the amylase from Halobacterium halobium . J Bacteriol 104:601–603
    [Google Scholar]
  11. Grant W. D., Gemmell R. T., McGenity T. J. 1998; Halophiles. In Extremophiles: Microbial Life in Extreme Environments pp 93–132 Edited by Horikoshi K., Grant W. D. New York: Wiley;
    [Google Scholar]
  12. Horinouchi S., Fukisumi S., Oshima T., Beppu T. 1988; Cloning and expression in Escherichia coli of two additional amylases of a strictly anaerobic thermophile, Dictyoglomus thermophilum , and their nucleotide sequences with extremely low guanine-plus-cytosine contents. Eur J Biochem 176:243–253 [CrossRef]
    [Google Scholar]
  13. Jana M., Chattopadhyay D. J., Pati B. R. 1997; Thermostable, high salt-tolerant amylase from Bacillus megaterium VUMB-109. Acta Biochim Biophys Hung 44:281–289
    [Google Scholar]
  14. Kobayashi T., Kanai H., Hayashi T., Akiba T., Akaboshi R., Horikoshi K. 1992; Haloalkaliphilic maltotriose-forming α-amylase from the archaebacterium Natronococcus sp. strain Ah-36. J Bacteriol 174:3439–3444
    [Google Scholar]
  15. 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
    [Google Scholar]
  16. Liebl W., Feil R., Gabelsberger J., Kellermann J., Schleifer K.-H. 1992; Purification and characterization of a novel thermostable 4-α-glucanotransferase of Thermotoga maritima cloned in Escherichia coli . Eur J Biochem 207:81–88 [CrossRef]
    [Google Scholar]
  17. Liebl W., Stemplinger I., Ruile P. 1997; Properties and gene structure of the Thermotoga maritima α-amylase amyA , a putative lipoprotein of a hyperthermophilic bacterium. J Bacteriol 179:941–948
    [Google Scholar]
  18. Metz R. J., Allen L. N., Cao T. M., Zeman N. W. 1988; Nucleotide sequence of an amylase gene from Bacillus megaterium . Nucleic Acids Res 16:5203 [CrossRef]
    [Google Scholar]
  19. Mijts B. N., Patel B. K. C. 2001; Random sequence analysis of genomic DNA of an anaerobic, thermophilic, halophilic bacterium, Halothermothrix orenii . Extremophiles 5:61–69 [CrossRef]
    [Google Scholar]
  20. Nakajima R., Imanaka T., Aiba S. 1986; Comparison of amino acid sequences of eleven different α-amylases. Appl Microbiol Biotechnol 23:355–360
    [Google Scholar]
  21. Nielsen H., Engelbrecht J., Brunak S., von Heijne G. 1997; A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Int J Neural Syst 8:581–599 [CrossRef]
    [Google Scholar]
  22. Onishi H., Sonoda K. 1979; Purification and some properties of an extracellular amylase from a moderate halophile, Micrococcus halobius . Appl Environ Microbiol 38:616–620
    [Google Scholar]
  23. Violet M., Meunier J. C. 1989; Kinetic study of the irreversible thermal denaturation of Bacillus licheniformis α-amylase. Biochem J 263:665–670
    [Google Scholar]
  24. Yebra M. J., Blasco A., Sanz P. 1999; Expression and secretion of Bacillus polymyxa neopullulanase in Saccharomyces cerevisiae . FEMS Microbiol Lett 170:41–49 [CrossRef]
    [Google Scholar]
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