1992 Fiscal Year Final Research Report Summary
ANALYSIS OF OXIDATIVE FERMENTATION, BIOCHEMICAL AND GENE-TECHNOLOGICAL RECONSTITUTION OF CN-INSENSITIVE RESPIRATORY CHAIN OF ACETIC ACID BACTERIA
| Project/Area Number |
03660118
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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 |
応用微生物学・発酵学
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| Research Institution | Kansai University |
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Principal Investigator |
AMEYAMA Minoru Kansai Univ., Faculty of Eng., Biotech., Prof., 工学部, 教授 (90022053)
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| Co-Investigator(Kenkyū-buntansha) |
MATSUSHITA Kazunobu Yamaguchi Univ., Facul.Agr., Associate Prof., 農学部, 助教授 (50107736)
OIKAWA Tadao Kansai Univ., Faculty of Eng., Biotech., Assistant, 工学部, 助手 (80233005)
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| Project Period (FY) |
1991 – 1992
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| Keywords | Oxidative fermentation / Cyanide-insensitive respiratory chain / Sugar-oxidizing dehydrogenases / Electron transport system / Cytochrome o / Acetic acid bacteria / Artificial membrane vesicle |
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| Research Abstract |
1) Sugar oxidizing dehydrogenases have been investigated widely onstrains of acetic acid bacteria, genus Pseudomanas, and methylotrophic bacteria. These enzymes have been purified and characterized to having as the prosthetic groups PQQ or covalently bound flavin.
2) The ethanol oxidase respiratoruy chain of Gluconobacter suboxydans was characterized by using G.suboxydans subsp. alpha , a variant species of G.suboxydans incapable of oxidizing ethanol. The organism is known to be defective in the second subunit of allochol dehydrogenase (ADH) so that the membranes of the strain exhibit neither ADH nor ethanol oxidase activity. The enzyme activities have been shown to be restored by reconstituting the second subunit to the membranes. The second subunit of ADH has been shown to be a cytochrome c-553(CO).
3) Membrane-bound, pyrroloquinoline quinone-dependent, alcohol dehydrogenase functions as the primary dehydrogenase in the respiratory chain of acetic acid bacteria. In this study, an abili
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ty of the enzyme to derectly react with ubiquinone was investigated in alcohol dehuydrogenases purified from both A.aceti and G.suboxydans. It has been shown that alcohol dehydrogenase of acetic acid bacteria donates electrons directly to ubiquinone in the cytoplasmic membranes and thus the ethanol oxidase respiratory chain of acetic acid bacteria is constituted of only three membranous respiratory component, alcohol dehydrogenase, ubiquinone, and terminal ubiquinol oxidase.
4) A plasmid, pGEACl, carrying the cytochrome c-553(CO) gene was introduced in G.suboxydans subsp. alpha and could increase dehydrogenase activities for D-glucose, D-sorbitol and glycerol in the membranes. Oxidase activities for glucose, D-sorbitol and glycerol were also increasedin both the membrane and the cells of G.suboxydans subsp.alpha haboring pGEACl. Furthermore, pGEACl could restore azide insensitivity in the respiratory chain of G.suboxydans subsp.alpha which lacks insensitivity against azide or cyanide. Thazide insensitivity was observed not only in the membranes but also in the intact cells of the organism(pGEACl). Thus, cytochrome c-553 (CO) seems to be indispensable in the pathway of the azide-insensitive respriratory chain bypass of G.suboxydans. In addition, the increase of oxidative ability in the practical oxidative fermentation was observed in the intact cells of G.suboxydans subsp.alpha (PGEACl) which increased the azide insensitivity in the respiratoy chain. Less
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