Molecular cloning and functional expression of a new amylosucrase from Alteromonas macleodii.
The presence of amylosucrase in 12 Alteromonas and Pseudoalteromonas strains was examined. Two Alteromonas species (Alteromonas addita KCTC 12195 and Alteromonas macleodii KCTC 2957) possessed genes that had high sequence homology to known amylosucrases. Genomic clones containing the ASase analogs were obtained from A. addita and A. macleodii, and the deduced amino acid sequences of the corresponding genes (aaas and amas, respectively) revealed that they were highly similar to the ASases of Neisseria polysaccharea, Deinococcus radiodurans, and Deinococcus geothermalis. Functional expression of amas in Escherichia coli was successful, and typical ASase activity was detected in purified recombinant AMAS, whereas the purified recombinant AAAS was nonfunctional. Although maximum total activity of AMAS was observed at 45 degrees C, the ratio of transglycosylation to total activity increased as the temperature decreased from 55 to 25 degrees C. These results imply that transglycosylation occurs preferentially at lower temperatures while hydrolysis is predominant at higher temperatures.
Ha
Suk-Jin
SJ
Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Yongin, Korea.
Seo
Dong-Ho
DH
Jung
Jong-Hyun
JH
Cha
Jaeho
J
Kim
Tae-Jip
TJ
Kim
Young-Wan
YW
Park
Cheon-Seok
CS
D020591:Alteromonas / Q000201:enzymology*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005964:Glucosyltransferases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010802:Phylogeny; D011108:Polymers / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013696:Temperature
D011108:Polymers; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C019700:amylosucrase