title: Biochemical characterization of a novel cycloisomaltooligosaccharide glucanotransferase from Paenibacillus sp. 598K.
abstract: Cycloisomaltooligosaccharide glucanotransferase (CITase; EC 2.4.1.248), a member of the glycoside hydrolase family 66 (GH66), catalyzes the intramolecular transglucosylation of dextran to produce cycloisomaltooligosaccharides (CIs; cyclodextrans) of varying lengths. Eight CI-producing bacteria have been found; however, CITase from Bacillus circulans T-3040 (CITase-T3040) is the only CI-producing enzyme that has been characterized to date. In this study, we report the gene cloning, enzyme characterization, and analysis of essential Asp and Glu residues of a novel CITase from Paenibacillus sp. 598K (CITase-598K). The cit genes from T-3040 and 598K strains were expressed recombinantly, and the properties of Escherichia coli recombinant enzymes were compared. The two CITases exhibited high primary amino acid sequence identity (67%). The major product of CITase-598K was cycloisomaltoheptaose (CI-7), whereas that of CITase-T3040 was cycloisomaltooctaose (CI-8). Some of the properties of CITase-598K are more favorable for practical use compared with CITase-T3040, i.e., the thermal stability for CITase-598K (≤50°C) was 10°C higher than that for CITase-T3040 (≤40°C); the k(cat)/K(M) value of CITase-598K was approximately two times higher (32.2s(-1)mM(-1)) than that of CITase-T3040 (17.8s(-1)mM(-1)). Isomaltotetraose was the smallest substrate for both CITases. When isomaltoheptaose or smaller substrates were used, a lag time was observed before the intramolecular transglucosylation reaction began. As substrate length increased, the lag time shortened. Catalytically important residues of CITase-598K were predicted to be Asp144, Asp269, and Glu341. These findings will serve as a basis for understanding the reaction mechanism and substrate recognition of GH66 enzymes.
authors.affiliation: National Agriculture and Food Research Organization, Kannondai, Tsukuba, Japan.
authors.forename: Ryuichiro
authors.initials: R
authors.lastname: Suzuki
authors.forename: Kazue
authors.initials: K
authors.lastname: Terasawa
authors.forename: Keitarou
authors.initials: K
authors.lastname: Kimura
authors.forename: Zui
authors.initials: Z
authors.lastname: Fujimoto
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Momma
authors.forename: Mikihiko
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Atsuo
authors.initials: A
authors.lastname: Kimura
authors.forename: Kazumi
authors.initials: K
authors.lastname: Funane
mesh_terms: D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry* / Q000235:genetics; D001407:Bacillus / Q000737:chemistry* / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003505:Cyclodextrins / Q000096:biosynthesis; D003911:Dextrans / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics; D006358:Hot Temperature; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D056507:Paenibacillus / Q000737:chemistry* / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D003505:Cyclodextrins; D003911:Dextrans; D007527:Isoenzymes; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D005964:Glucosyltransferases; C087517:cycloisomaltooligosaccharide glucanotransferase