1994 Fiscal Year Final Research Report Summary
Characterization and structure of extremely thermostable oxidoreductases from anaerobic hyperthermophilic archaea
| Project/Area Number |
05808055
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
Structural biochemistry
|
|---|
| Research Institution | Kyoto university of Education |
|---|
Principal Investigator |
OHSHIMA Toshihisa Department of Chemistry, Kyoto University of Education, Professor, 教育学部, 教授 (10093345)
|
|---|
| Project Period (FY) |
1993 – 1994
|
| Keywords | Anaerobic hyperthermophilic archae / extremely thermostable enzyme / Pyrobaculum islandicum / Glutamate dehydrogenase |
|---|
| Research Abstract |
During the past decade, many anaerobic hyperthermophiles growing at temperatures near or above 100ﾟC have been isolated from marine and continental volcanic environments. Rapidly expanding interests have been shown in hyperthermophiles. In particular, most interest is focused on understanding the adaptation mechanisms that allow metabolism to function and biomolecules such as protein, enzyme and DNA to remain intact at extremely high temperatures. Most hyperthermophiles are belong to the archaea, the third domain of life, and evolutionary attentions have been paid on their biomolecules. In addition, enzymes from the hyperthermophiles have large biotechnological potential.
In this study, we purified NAD-dependent GluDH (EC 1.4.1.2) from a continental hyperthermophilic archaeon Pyrobaculum islandicum, and the enzyme was characterized by comparison with those from other hyperthermophiles.
The enzyme purified to homogeneity from a thermophilic archaeon, P.islandicum by two successive Red-Sepharose CL-4B affinity chromatographies has a molecular mass of about 220 kDa and consists of six subunits with identical molecular masses of 36 kDa. The enzyme is extremely thermostable ; the activity is not lost after incubation at 100ﾟC for 2 h. The enzyme activity increased linearly with temperature and the maximum was observed around 90ﾟC.The enzyme acts on L-norvaline, L-2-aminobutyrate and L-valine as well as L-glutamatefor the deamination in the presence of NAD.For reductive amination, 2-oxoglutarate was the most preferable substrate in the presence of NADH.The Km values for NAD,L-glutamate, NADH,2-oxoglutarate and ammonia were 0.26,0.11,0.044,0.13 and 3.6mM,respectively. The enzyme activity was increased by the addition of denaturants such as guanidine-HCl and some water-soluble organic solvents such as ethanol and toluene. These properties are different from those of other NADP-dependent GluDHs of marine hyperthermophiles.
|
Research Products
(2 results)
