Extracellular synthesis, specific recognition, and intracellular degradation of cyclomaltodextrins by the hyperthermophilic archaeon Thermococcus sp. strain B1001.
A unique extracellular and thermostable cyclomaltodextrin glucanotransferase (CGTase) from the hyperthermophilic archaeon Thermococcus sp. strain B1001 produces predominantly (>85%) alpha-cyclomaltodextrin (alpha-CD) from starch (Y. Tachibana, et al., Appl. Environ. Microbiol. 65:1991--1997, 1999). Nucleotide sequencing of the CGTase gene (cgtA) and its flanking region was performed, and a cluster of five genes was found, including a gene homolog encoding a cyclomaltodextrinase (CDase) involved in the degradation of CDs (cgtB), the gene encoding CGTase (cgtA), a gene homolog for a CD-binding protein (CBP) (cgtC), and a putative CBP-dependent ABC transporter involved in uptake of CDs (cgtDE). The CDase was expressed in Escherichia coli and purified. The optimum pH and temperature for CD hydrolysis were 5.5 and 95 degrees C, respectively. The molecular weight of the recombinant enzyme was estimated to be 79,000. The CDase hydrolyzed beta-CD most efficiently among other CDs. Maltose and pullulan were not utilized as substrates. Linear maltodextrins with a small glucose unit were very slowly hydrolyzed, and starch was hydrolyzed more slowly. Analysis by thin-layer chromatography revealed that glucose and maltose were produced as end products. The purified recombinant CBP bound to maltose as well as to alpha-CD. However, the CBP exhibited higher thermostability in the presence of alpha-CD. These results suggested that strain B1001 possesses a unique metabolic pathway that includes extracellular synthesis, transmembrane uptake, and intracellular degradation of CDs in starch utilization. Potential advantages of this starch metabolic pathway via CDs are discussed.
Hashimoto
Y
Y
CREST, Osaka, Japan.
Yamamoto
T
T
Fujiwara
S
S
Takagi
M
M
Imanaka
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D002855:Chromatography, Thin Layer; D004269:DNA, Bacterial / Q000737:chemistry; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011134:Polysaccharides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D013213:Starch / Q000378:metabolism; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology / Q000378:metabolism*
D004269:DNA, Bacterial; D011134:Polysaccharides; D011994:Recombinant Proteins; C008315:maltodextrin; D013213:Starch; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; D006026:Glycoside Hydrolases; C048745:cyclomaltodextrinase