Applied Microbiology and Biotechnology

, Volume 86, Issue 2, pp 555–566 | Cite as

Characterization of an exo-acting intracellular α-amylase from the hyperthermophilic bacterium Thermotoga neapolitana

  • Kyung-Min Park
  • So-Young Jun
  • Kyoung-Hwa Choi
  • Kwan-Hwa Park
  • Cheon-Seok Park
  • Jaeho Cha
Biotechnologically Relevant Enzymes and Proteins

Abstract

We cloned and expressed the gene for an intracellular α-amylase, designated AmyB, from the hyperthermophilic bacterium Thermotoga neapolitana in Escherichia coli. The putative intracellular amylolytic enzyme contained four regions that are highly conserved among glycoside hydrolase family (GH) 13 α-amylases. AmyB exhibited maximum activity at pH 6.5 and 75°C, and its thermostability was slightly enhanced by Ca2+. However, Ca2+ was not required for the activity of AmyB as EDTA had no effect on enzyme activity. AmyB hydrolyzed the typical substrates for α-amylase, including soluble starch, amylose, amylopectin, and glycogen, to liberate maltose and minor amount of glucose. The hydrolytic pattern of AmyB is most similar to those of maltogenic amylases (EC 3.2.1.133) among GH 13 α-amylases; however, it can be distinguished by its inability to hydrolyze pullulan and β-cyclodextrin. AmyB enzymatic activity was negligible when acarbose, a maltotetraose analog in which a maltose residue at the nonreducing end was replaced by acarviosine, was present, indicating that AmyB cleaves maltose units from the nonreducing end of maltooligosaccharides. These results indicate that AmyB is a new type exo-acting intracellular α-amylase possessing distinct characteristics that distinguish it from typical α-amylase and cyclodextrin-/pullulan-hydrolyzing enzymes.

Keywords

GH 13 α-amylases Hyperthermophiles Intracellular α-amylase Thermostability Thermotoga neapolitana 

Notes

Acknowledgments

This study was supported, in part, by the Marine and Extreme Genome Research Center Program of the Ministry of Land, Transportation and Maritime Affairs and by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD; KRF-2007-521-F00056), Republic of Korea.

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Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Kyung-Min Park
    • 1
  • So-Young Jun
    • 1
  • Kyoung-Hwa Choi
    • 1
  • Kwan-Hwa Park
    • 2
  • Cheon-Seok Park
    • 3
  • Jaeho Cha
    • 1
  1. 1.Department of Microbiology, College of Natural SciencesPusan National UniversityBusanKorea
  2. 2.Department of BiologyUniversity of IncheonIncheonKorea
  3. 3.Graduate School of Biotechnology, and Institute of Life Science and ResourcesKyung Hee UniversityYonginKorea

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