Molecular cloning and characterization of a novel gamma-CGTase from alkalophilic Bacillus sp.
We found a novel cyclodextrin glucanotransferase (CGTase) from alkalophilic Bacillus sp. G-825-6. The enzyme was expressed in the culture broth by recombinant Bacillus subtilis KN2 and was purified and characterized. The enzyme named CGTase825-6 showed 95% amino acid sequence identity with a known enzyme beta-/gamma-CGTase from Bacillus firmus/lentus 290-3. However, the product specificity of CGTase825-6 differed from that of beta-/gamma-CGTase. CGTase825-6 produced gamma-cyclodextrin (CD) as the main product, but degradation of gamma-CD was observed with prolonged reaction. The product specificity of the enzyme was positioned between gamma-CGTase produced by Bacillus clarkii 7364 and B. firmus/lentus 290-3 beta-/gamma-CGTase. It showed that the difference of product specificity was dependent on only 28 amino acid residues in 671 residues in CGTase825-6. We compared the amino acid sequence of CGTase825-6 and those of other CGTases and constructed a protein structure model of CGTase825-6. The comparison suggested that the diminished loop (Val138-Asp142) should provide subsite -8 for gamma-CD production and that Asp142 might have an important role in product specificity. CGTase825-6 should be a useful tool to produce gamma-CD and to study the differences of producing mechanisms between gamma-CD and beta-CD.
Hirano
Kyoko
K
Laboratory of Molecular Enzymology, Division of Life Science, Graduate School of Agricultural Science, Tohoku University, Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, Japan.
Ishihara
Takeo
T
Ogasawara
Satoshi
S
Maeda
Hiroshi
H
Abe
Keietsu
K
Nakajima
Tasuku
T
Yamagata
Youhei
Y
D000595:Amino Acid Sequence; D001407:Bacillus / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D047408:gamma-Cyclodextrins / Q000378:metabolism*
D011994:Recombinant Proteins; D047408:gamma-Cyclodextrins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase