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Journal of Biochemistry Advance Access published online on July 21, 2006
Journal of Biochemistry, doi:10.1093/jb/mvj158
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© 2006 The Japanese Biochemical Society.
Received December 19, 2005
Accepted June 4, 2006

Regular Paper

Site-Directed Mutations in Alanine 223 and Glycine 255 in the Acceptor Site of {gamma}-Cyclodextrin Glucanotransferase from Alkalophilic Bacillus clarkii 7364 Affect Cyclodextrin Production

Yoshinori Nakagawa 1 *, Masayasu Takada 2, Koichi Ogawa 2, Yuji Hatada 3, and Koki Horikoshi 3

1 Nihon Shokuhin Kako Co., Ltd, 30 Tajima, Fuji 417-8530, Japan; Extremobiosphere Research Center, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima, Yokosuka 237-0061, Japan
2 Nihon Shokuhin Kako Co., Ltd, 30 Tajima, Fuji 417-8530, Japan
3 Extremobiosphere Research Center, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima, Yokosuka 237-0061, Japan

* To whom correspondence should be addressed.
Yoshinori Nakagawa, E-mail: yoshinori.nakagawa{at}jamstec.go.jp


  Abstract

A cyclodextrin glucanotransferase (CGTase) from Bacillus clarkii 7364 converts starch into {gamma}-cyclodextrin ({gamma}-CD) with high specificity. Comparison of the deduced amino acid sequence of this CGTase with those of other typical CGTases revealed that several amino acids are deleted or substituted with others at several subsites. Of these amino acids, Ala223 at subsite +2 and Gly255 at subsite +3 in the acceptor site of the enzyme were replaced by several amino acids through site-directed mutagenesis. The replacement of Ala223 by lysine, arginine and histidine strongly enhanced the {gamma}-CD-forming activity in the neutral pH range. On the other hand, all mutants obtained on replacing Gly255 with the above amino acids showed significant decreases in the {gamma}-CD-forming activity. Taking into account both the kinetic parameters and pKa values of the side chains of the three basic amino acids, the protonation state of the amino groups in their side chains at subsite +2 seems to enhance the hydrogen bonding interaction between these basic amino acids and the glucose residues of linear oligosaccharides. The enhancement of the interaction may play an important role by helping the substrate reach subsite +1, hence increasing the {gamma}-CD-forming activity and kcat value.

Keywords: acceptor site; cyclodextrin; {gamma}-CD-forming activity; {gamma}-CGTase; site-directed mutagenesis.
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