Biochemical characterization of a novel cycloisomaltooligosaccharide glucanotransferase from Paenibacillus sp. 598K |
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Authors: | Ryuichiro Suzuki Kazue Terasawa Keitarou Kimura Zui Fujimoto Mitsuru Momma Mikihiko Kobayashi Atsuo Kimura Kazumi Funane |
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Institution: | 1. Applied Microbiology Division, National Food Research Institute, National Agriculture and Food Research Organization, 2-1-12 Kannondai, Tsukuba 305-8642, Japan;2. Biomolecular Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba 305-8602, Japan;3. Department of Food and Health Science, Jissen Women''s University, 4-1-1 Osakaue, Hino 191-8510, Japan;4. Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Kita-9 Nisi-9, Kita-ku, Sapporo 060-8589, Japan |
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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 kcat/KM value of CITase-598K was approximately two times higher (32.2 s− 1 mM− 1) than that of CITase-T3040 (17.8 s− 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. |
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Keywords: | CBM carbohydrate-binding module CI cycloisomaltooligosaccharide CITase CI glucanotransferase CITase-598K CITase from Paenibacillus sp 598K CITase-T3040 CITase from Bacillus circulans T-3040 DP degree of polymerization EDTA ethylenediaminetetraacetic acid EGTA ethylene glycol tetraacetic acid GH glycoside hydrolase HPLC high-performance liquid chromatography IG isomaltooligosaccharide IG2 isomaltose IG3 isomaltotriose IG4 isomaltotetraose IG5 isomaltopentaose IG6 isomaltohexaose IG7 isomaltoheptaose PCR polymerase chain reaction PsDex dextranase from Paenibacillus sp SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis SmuDXA dextranase from Streptococcus mutans |
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