| Site-directed mutations in Alanine 223 and Glycine 255 in the acceptor site of gamma-Cyclodextrin glucanotransferase from Alkalophilic Bacillus clarkii 7364 affect cyclodextrin production. | |
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MedLine Citation:
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PMID: 16861250 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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A cyclodextrin glucanotransferase (CGTase) from Bacillus clarkii 7364 converts starch into gamma-cyclodextrin (gamma-CD) with high specificity. Comparison of the deduced amino acid sequence of this CGTase with those of other typical CGTases revealed that several amino acids are deleted or substituted with others at several subsites. Of these amino acids, Ala223 at subsite +2 and Gly255 at subsite +3 in the acceptor site of the enzyme were replaced by several amino acids through site-directed mutagenesis. The replacement of Ala223 by lysine, arginine and histidine strongly enhanced the gamma-CD-forming activity in the neutral pH range. On the other hand, all mutants obtained on replacing Gly255 with the above amino acids showed significant decreases in the gamma-CD-forming activity. Taking into account both the kinetic parameters and pKa values of the side chains of the three basic amino acids, the protonation state of the amino groups in their side chains at subsite +2 seems to enhance the hydrogen bonding interaction between these basic amino acids and the glucose residues of linear oligosaccharides. The enhancement of the interaction may play an important role by helping the substrate reach subsite +1, hence increasing the gamma-CD-forming activity and kcat value. |
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Authors:
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Yoshinori Nakagawa; Masayasu Takada; Koichi Ogawa; Yuji Hatada; Koki Horikoshi |
Publication Detail:
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Type: Comparative Study; Journal Article Date: 2006-07-21 |
Journal Detail:
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Title: Journal of biochemistry Volume: 140 ISSN: 0021-924X ISO Abbreviation: J. Biochem. Publication Date: 2006 Sep |
Date Detail:
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Created Date: 2006-09-18 Completed Date: 2007-01-16 Revised Date: 2007-12-19 |
Medline Journal Info:
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Nlm Unique ID: 0376600 Medline TA: J Biochem Country: Japan |
Other Details:
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Languages: eng Pagination: 329-36 Citation Subset: IM |
Affiliation:
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Nihon Shokuhin Kako Co., Ltd., 30 Tajima, Fuji 417-8530. yoshinori.nakagawa@jamstec.go.jp |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acid Sequence Bacillus / enzymology*, genetics Base Sequence DNA Primers Glucosyltransferases / chemistry, genetics* Molecular Sequence Data Mutagenesis, Site-Directed Protein Conformation Sequence Analysis, DNA Species Specificity gamma-Cyclodextrins / metabolism* |
| Chemical | |
Reg. No./Substance:
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0/DNA Primers; 0/gamma-Cyclodextrins; 17465-86-0/gamma-cyclodextrin; EC 2.4.1.-/Glucosyltransferases; EC 2.4.1.19/cyclomaltodextrin glucanotransferase |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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