Paenibacillus sp. A11 produced an intracellular cyclodextrinase (CDase), its presence was confirmed by activity detection on an agar plate with specific screening medium containing P-cyclodextrin (P-CD) and phenol phthalein. The CDase was purified up to 22-fold with a 28% yield. The enzyme was a single polypeptide with a molecular weight of 80 kDa. Optimum activity was at pH 7.0 and 40degreesC. The enzyme had an isoelectric point of 5.4 and N-terminal sequence was M F L E A V Y H R P R K N W S. When relative hydrolytic activities of the CDase on different substrates were compared, it was found that high specificity was exerted by P-CD while maltoheptaose, its linear counterpart, was only 40% as active. The enzyme recognized alpha-1,4-glucose units and the hydrolysis depended on the size of oligosaccharides. Highly branched carbohydrates such as glycogen or dextran or other heteropolymers as glucomannan could not be hydrolyzed. This enzyme was different from other CDases in its ability to hydrolyze maltose and trehalose, though with very low hydrolytic activity. The major product from all substrates was maltose. The k(cat)/K-m value for beta-CD was 8.28 x 10(5) M-1 min(-1). The enzyme activity was completely inactivated by 1 mM N-bromosuccinimide and diethylpyrocarbonate suggesting the crucial importance of Trp and His for its catalytic activity. Essential Trp was confirmed to be at enzyme active site by substrate protection experiment. Partial inactivation by 5 mM phenylglyoxal suggests the involvement of Arg, which has never been reported in other CDases. (C) 2004 Elsevier Inc. All rights reserved.