1994 Fiscal Year Final Research Report Summary
Spectroscopic and Kinetic Studies on the Active Site Structure and Substrate Specificities of Pullulanase.
| Project/Area Number |
04660097
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
物理計測・光学
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| Research Institution | Fukuyama University |
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Principal Investigator |
HIROMI Keitaro Fukuyama University, Faculty of Engineering, Professor, 工学部, 教授 (50025425)
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| Co-Investigator(Kenkyū-buntansha) |
MUROOKA Yoshikatsu Hiroshima University, Faculty of Engineering, Professor, 工学部, 教授 (60029882)
IWAMOTO Hiroyuki Fukuyama University, Faculty of Engineering, Lecturer, 工学部, 講師 (90213321)
HIROSE Junzo Fukuyama University, Faculty of Engineering, Associate Professor, 工学部, 助教授 (70080215)
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| Project Period (FY) |
1992 – 1994
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| Keywords | pullulanase / cyclodextrins / active site of enzyme |
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| Research Abstract |
1) In the interaction of cyclodextrins (CDs) with pullulanase from Klebsiella pneumoniae, it has been known that beta-CD has the highest affinity compared with alpha- and gamma-CDs. Two lines of approach have strongly indicated that some side chain of the enzyme is included in only beta-CD ring. First, mono-tosylated beta-CD among the three CDs has the most decreased affinity. Second, adamantan carboxylate which is known to be included in the ring of beta-CD most remarkably decreased the binding affinity.
2) Pullulanase from Klebsiella aerogenes has been found to have significantly different (better) K_m and k_<cat> values and also much stronger inhibition by cyclodextrins. Moreover, the shape of the difference fluorescence spectra caused by pullulan and cyclodextrins are significantly different from those of the enzyme from Klebsiella pneumoniae.
3) Several kinds of site-directed mutagenesis of pullulanase from Klebsiella aerogenes were successfully done to determine the roles of amono acid residues importantly involved in the enzyme reaction.
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