Characterization of two gamma-cyclodextrin-specific enzymes from Bacillus clarkii 7364
2010
Nakagawa, Y., Nihon Shokuhin Kako Co. Ltd., Fuji, Shizuoka (Japan) | Saburi, W. | Yamamoto, T. | Takada, M. | Ogawa, K. | Yamamoto, M. | Hatada, Y. | Nakamura, N. | Horikoshi, K.
The alkaliphilic soil bacterium Bacillus clarkii 7364 was found to produce cyclodextrin glucanotransferase (CGTase), an enzyme which converts starch into gamma-cyclodextrin (gamma-CD) with high specificity. The bacterium also intracellularly produced cyclodextrinase (CDase). The gene encoding the CDase (Cda) was located about 200 bases downstream of the gene encoding the CGTase (Cgt). Comparison of the amino acid sequence of Cgt with those of other CGTases revealed that several amino acids which contribute to substrate binding are absent or different at subsites +3, +2, -3 and -7 in Cgt. The replacement of Ala223 at subsite +2 by three basic amino acids, histidine, lysine and arginine, strongly enhanced gamma-CD-forming activity in the neutral pH range, although the optimum pH for the activity (pH 10.0) and CD production specificity remained the same. The interaction between the protonated amino group in the side chain of these basic amino acids and the linear substrate is thought to play an important role in the enhancement of the activity. Cda had extremely high hydrolytic specificity for gamma-CD. Cda also displayed a transglycosylation activity, where a maltotriose moiety could be transferred, unlike other CD-degrading enzymes. In Cda, the N-domain normally found in other CD-degrading enzymes which functions in the dimerization, thus contributing to the preference for CDs, was deleted and instead, a long extra sequence was present in the C-terminus. Despite the lack of the N-domain, Cda showed a dodecameric structure. These unique features indicate that Cda is a novel CD-degrading enzyme.
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