Identification of Catalytic Amino Acids of Cyclodextran Glucanotransferase from Bacillus circulans T-3040
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- YAMAMOTO Tomoko
- National Food Research Institute
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- TERASAWA Kazue
- National Food Research Institute
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- KIM Young-Min
- Laboratory of Molecular Enzymology, Hokkaido University
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- KIMURA Atsuo
- Laboratory of Molecular Enzymology, Hokkaido University
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- KITAMURA Yoshiaki
- National Food Research Institute
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- KOBAYASHI Mikihiko
- Jissen Women’s University
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- FUNANE Kazumi
- National Food Research Institute
Bibliographic Information
- Other Title
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- Identification of Catalytic Amino Acids of Cyclodextran Glucanotransferase from<i>Bacillus circulans</i>T-3040
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Abstract
In glycoside hydrolase family 66 (see http://afmb.cnrs-mrs.fr/CAZY/), cyclodextran glucanotransferase (CITase) is the only transglycosylation enzyme, all the other family 66 enzymes being dextranases. To analyze the catalytic amino acids of CITase, we modified CITase chemically from the T-3040 strain of Bacillus circulans with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). EDC inactivated the enzyme by following pseudo-first order kinetics. In addition, the substrates of an isomaltooligosaccharide and a cyclodextran inhibited EDC-induced enzyme inactivation, implicating the carboxyl groups of CITase as the catalytic amino acids of the enzyme. When two conserved aspartic acid residues, Asp145 and Asp270, were replaced with Asn in T-3040 mature CITase, CIT-D270N was completely inactive, and CIT-D145N had reduced activity. The Vmax of CIT-D145N was 1% of that of wild-type CITase, whereas the Km of CIT-D145N was about the same as that of the wild-type enzyme. These findings indicate that Asp145 and Asp270 play an important role in the enzymatic reaction of T-3040 CITase.
Journal
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- Bioscience, Biotechnology, and Biochemistry
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Bioscience, Biotechnology, and Biochemistry 70 (8), 1947-1953, 2006
Japan Society for Bioscience, Biotechnology, and Agrochemistry
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Details 詳細情報について
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- CRID
- 1390001206479478144
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- NII Article ID
- 10018528285
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- NII Book ID
- AA10824164
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- ISSN
- 13476947
- 09168451
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- NDL BIB ID
- 8040467
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- Text Lang
- en
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- Data Source
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- JaLC
- NDL
- Crossref
- CiNii Articles
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- Abstract License Flag
- Disallowed