1990 Fiscal Year Final Research Report Summary
Stabilization of Glucose Dehydrogenase by Strenghthening Intersubunit Association
| Project/Area Number |
01550762
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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 |
発酵工学
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| Research Institution | Osaka University |
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Principal Investigator |
URABE Itaru Osaka University, Dept. of Ferment. Technol., Assoc. Professor, 工学部, 助教授 (60029246)
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| Project Period (FY) |
1989 – 1990
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| Keywords | Glucose dehydrogenase / Random mutagenesis / Amino acid substitution / Thermo-stability / アミノ酸置換 |
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| Research Abstract |
Glucose dehydrogenase (GDH) gene was isolated from Bacillus megaterium IWG3, and the gene was mutagenized with hydrazine, formic acid, or sodium nitrite, and 12 clones containing mutant genes for thermostable GDH were obtained. The nucleotide sequences of the 12 genes show that they include 8 kinds of mutants having the following amino acid substitutions : Glu-96 to Gly ; Glu-96 to Ala ; Gln-252 to Leu ; Gln-252 to Leu and Ala-258 to Gly ; Glu-96 to Lys and Val-183 to Ile ; Glu-96 to Lys, Val-112 to Ala, Glu-133 to Lys, and Tyr-217 to His ; glu-96 to Lys, Asp-108 to Asn. Pro-194 to Gln, and Glu-210 to Lys ; Tyr-253 to Cys. These mutant enzymes have higher stability at 60^ﾟC than the wild-type enzyme. The results of this study indicate that tetrameric structure of GDH is stabilized by several kinds of mutation, and at least one of the following amino asid substitutions stabilizes the enzyme : Glu-96 to Gly, Glu-96 to Ala, Gln-252 to Leu, and Tyr-253 to Cys.
These four stability-increasing mutants of GDH were purified together with the wild-type enzyme. These mutant enzymes are more heat resistant at pH 6.5 than the wild-type enzyme ; the replacement of Glu-96 by Ala increases the thermostability by about 20^ﾟC. The mutant enzymes also resistant to inactivation in alkaline solutions. The replacement of Glu-96 by Gly or Ala protects the enzyme from alkaline inactivation almost completely. The kinetic constants for the activities of these mutant enzymes do not differ significantly from those of wild-type enzyme.
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Research Products
(6 results)
