Novel cyclodextrin glucanotransferase, process for producing the same and process for producing cyclodextrin by using this enzyme

#The present invention relates to cyclodextrin glucanotransferase (EC 2.4.1.19, referred to as “CGTase” hereinafter), a method of manufacturing the same, and a method of manufacturing cyclodextrin (referred to as “CD” hereinafter) employing the same.

#CGTase (EC 2.4.1.19) is an enzyme functioning on α-1,4-glucans such as starches to produce cyclodextrins (CD), which are cyclic α-1,4-glucans, through its intramolecular transfer activity. The degree of polymerization of the CDs produced by CGTase is chiefly 6-8, with these products being referred to as α-, β and γ-CD, respectively. In addition to this CD-producing reaction, CGTase catalyzes coupling reactions (the ring of the CD is opened and the resulting straight-chain oligosaccharide is transferred to a receptor sugar molecule) and disproportionation reactions (a straight-chain oligosaccharide is transferred to the receptor sugar molecule) through the intramolecular transfer reaction. Further, albeit weakly, CGTase also catalyzes the hydrolysis reaction of the α-1,4-glucoside bond.

#Since CDs can change chemical and physical properties of various molecules by making clathrates therewith, CGTase has achieved a position as an important enzyme in the food, pharmaceutical and cosmetic industries. Thus, beginning from the CD synthesis reaction by the Bacillus macerans enzyme in 1939 (E. B. Tilden and S. J. Pirt, J. Am. Chem. Soc., 63, 2900-2902, 1939), a large number of studies were conducted, including a search for bacteria producing CGTase and means of purifying the enzyme (Sumio Kitahata, Naoto Tsuyama and Shigetaka Okada, Agr. Biol. Chem., 38 (2), 387-393, 1974; Sumio Kitahata and Shigetaka Okada, Agr. Biol. Chem., 38 (12), 2413-2417, 1974; Sumio Kitahata and Shigetaka Okada, J. Jap. Soc. Starch Sci., 29 (1), 13-18, 1982; Michio Kubota, Yoshiki Matsuura, Shuzo Sakai and Yukiteru Katsube, Denpun Kagaku, 38 (2), 141-146, 1991; Lionel J. Bovetto, Daniel P. Backer, Jaques R. Villette, Philippe J. Sicard, and Stephane J-L. Bouquelet, Biotechnology and Applied Biochemstry, 15, 48-58, 1992; Shinske Fujiwara, Hirofumi Kakihara, Kim Myung Woo, Andre Lejeune, Mitsuhide Kanemoto, Keiji Sakaguchi, and Tadayuki Imanaka, Applied and environmental microbiology, 58 (12), 4016-4025, 1992; Florian Binder, Otto Huber and August Bock, Gene, 47, 269-277, 1986; Keiji Kainuma, Toshiya Takano and Kunio Yamane, Appl. Microbiol. Biotechnol., 26, 149-153, 1987; Takahiro Kaneko, Tetsuo Hamamoto and Koki Horikoshi, J. general Microbiology, 134, 97-105, 1988; Murai Makela, Pekka Mattsson, M. Eugenia Schinina, and Timo Korpela, Biotechnology and Applied biochemistry, 10, 414-427, 1988; Ernest K. C. Yu, Hiroyuki Aoki, and Masanaru Misawa, Appl. Microbiol. Biotechnol., 28, 377-379, 1988).

#FIG. 1 is a graph showing the effect of pH on the activity of γ-CGTase derived from Bacillus clarkii 7364 (Blue value method).

#FIG. 2 is a graph showing the effect of temperature on the activity of γ-CGTase derived from Bacillus clarkii 7364 (Blue value method).

#FIG. 3 is a graph showing change over time in the γ-CD production reaction using soluble starch as a substrate.

#(Cyclodextrin Glucanotransferase)

#Table 1 gives the bacteriological properties of the strain newly discovered and separated by the present inventors. 1 TABLE 1 Various properties of the bacterial strain Culture temperature 37° C. Cell form Rod-shaped microorganism (0.6-0.8 × 3-5 μm) with elongated form Gram dyeing + Spores ± Motility + Colony form Shape of colony: irregular Perimeter of colony: filamentous Colony surface protrusions: low and flat Luster: None Color: Cream Size: Φ 3-4 mm Characteristics: Colonies adhere to medium Catalase + Oxidase + O/F Test − β-Galactosidase + Arginine hydrolase − Lysine decarboxylase − Ornithine decarboxylase − Use of citric acid − H2S production − Urease − Tryptophan deamidase − Indole production − Acetoin production + Gelatinase − Nitrate reduction − Hydrolysis Casein + Gelatin + Starch + Tween 20 − Tween 40 + Tween 60 + Phenyl alanine deamidase − Growth properties 10° C. − 40° C. + 50° C. − pH 7 − 5% NaCl + 10% NaCl + Identification result Bacillus clarkii

#The 16 S rDNA nucleotide sequence of this bacterial strain is shown as SEQUENCE I.D. 1 in the sequence listing.