A gene encoding a cyclodextrin glucanotransferase (CGTase) from Thermococcus kodakaraensis KOD1 (CGT Tk ) was identified and characterized. The gene (cgt Tk ) encoded a protein of 713 amino acid residues harboring the four conserved regions found in all members of the ␣-amylase family. However, the C-terminal domain corresponding to domain E of previously known CGTases displayed a completely distinct primary structure. In order to elucidate the catalytic function of the gene product, the recombinant enzyme was purified by anion-exchange chromatography, and its enzymatic properties were investigated. The enzyme displayed significant starch-degrading activity (750 U/mg of protein) with an optimal temperature and pH of 80°C and 5.5 to 6.0, respectively. The presence of Ca 2؉ enhanced the enzyme activity and elevated the optimum temperature to 85 to 90°C. With the addition of Ca 2؉ , the enzyme showed extreme thermostability, with almost no loss of enzymatic activity after 80 min at 85°C, and a half-life of 20 min at 100°C. CGT Tk could hydrolyze soluble starch and glycogen but failed to hydrolyze pullulan. Most importantly, although CGT Tk harbored a unique C-terminal domain, we found that the protein also exhibited significant CGTase activity, with ␤-cyclodextrin as the main product. In order to identify the involvement, if any, of the C-terminal region in the CGTase activity, we analyzed a truncated protein (CGT Tk ⌬C) with 23 C-terminal amino acid residues deleted. CGT Tk ⌬C displayed similar properties in terms of starch-binding activity, substrate specificity, and thermostability, but unexpectedly showed higher starch-degrading activity than the parental CGT Tk . In contrast, the cyclization activity of CGT Tk ⌬C was abolished. The results indicate that the presence of the structurally novel C-terminal domain is essential for CGT Tk to properly catalyze the cyclization reaction.

Pitrilysin from Escherichia coli was overproduced, purified, and analyzed for enzymatic activity using 14 peptides as a substrate. Pitrilysin cleaved all the peptides, except for two of the smallest, at a limited number of sites, but showed little amino acid specificity. It cleaved -endorphin (-EP) most effectively, with a K m value of 0.36 M and a k cat value of 750 min À1 . -EP consists of 31 residues and was predominantly cleaved by the enzyme at Lys 19 -Asn 20 . Kinetic analyses using a series of -EP derivatives with N and/or C-terminal truncations and with amino acid substitutions revealed that three hydrophobic residues (Leu 14 , Val 15 , and Leu 17 ) and the region 22-26 in -EP are responsible for high-affinity recognition by the enzyme. These two regions are located on the N-and C-terminal sides of the cleavage site in -EP, suggesting that the substrate binding pocket of pitrilysin spans its catalytic site.

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