Characterization of an exo-acting intracellular alpha-amylase from the hyperthermophilic bacterium Thermotoga neapolitana.
We cloned and expressed the gene for an intracellular alpha-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 alpha-amylases. AmyB exhibited maximum activity at pH 6.5 and 75 degrees 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 alpha-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 alpha-amylases; however, it can be distinguished by its inability to hydrolyze pullulan and beta-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 alpha-amylase possessing distinct characteristics that distinguish it from typical alpha-amylase and cyclodextrin-/pullulan-hydrolyzing enzymes.
Park
Kyung-Min
KM
Department of Microbiology, College of Natural Sciences, Pusan National University, San 30, Jangjeon-dong, Geumjeong-gu, Busan, 609-735, Korea.
Jun
So-Young
SY
Choi
Kyoung-Hwa
KH
Park
Kwan-Hwa
KH
Park
Cheon-Seok
CS
Cha
Jaeho
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D020536:Enzyme Activators; D004795:Enzyme Stability; D004926:Escherichia coli; D015870:Gene Expression; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D046949:Thermotoga neapolitana / Q000201:enzymology*; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D001426:Bacterial Proteins; D002413:Cations, Divalent; D004269:DNA, Bacterial; D020536:Enzyme Activators; D000516:alpha-Amylases; D002118:Calcium