| Project/Area Number |
14550764
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
生物・生体工学
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| Research Institution | Kitami Institute of Technology |
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Principal Investigator |
HORIUCHI Jun-ichi Kitami Institute of Technology, Faculty of Engineering, Professor, 工学部・化学システム工学科, 教授 (30301980)
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| Co-Investigator(Kenkyū-buntansha) |
TADA Kiyoshi Kitami Institute of Technology, Faculty of Engineering, Assistant, 工学部・化学システム工学科, 助手 (90333666)
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| Project Period (FY) |
2002 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2004: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2003: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2002: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Keywords | proteome analysis / 2-dimensional electrophoresis / lysine fermentation / fermentation phase / dynamic analysis of protein / 2次元電気泳動 / 最適化 / タンパク動態解析 / 前処理 |
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| Research Abstract |
The aim of this study is to develop a methodology to analyze and optimize bioprocesses based on intracellular information obtained by proteome analysis using a two-dimensional electrophoresis (2-DE). Threonine-limited lysine fermentation using Brevibacterium flavum was employed for the proteome analysis. Samples were taken from batch cultures of lysine fermentation at different physiological states including an exponential growth phase, an initial lysine production phase and a final lysine production phase. The cell-free extracts were obtained by homogenization using glass beads. The use of phenylmethylsulfonyl fluoride (protease inhibitor) and acetone precipitation greatly improved the extraction efficiency of intracellular proteins from intact cells. Approximately 480 and 450 protein spots were detected by 2-DE in the samples of the exponential growth phase and the initial lysine production phase, respectively. However, the number of protein spots decreased to approximately 320 in the sample of the final lysine production phase, which suggested the decline of microbial activity at the end of fermentation. The image analysis was then performed to identify protein spots of 2-DE maps by use of the database of Conrynebacterium glutamicum proteins analyzed by a peptide mass fingerprinting. At the exponential growth phase, proteins concerning glycolysis and TCA cycle were identified, while the proteins related to lysine biosynthesis was not observed. However, in the sample of the initial and final lysine production phases, 2 proteins (DapA and Doh) concerning lysine biosynthesis was clearly identified as well as proteins concerning glycolysis and TCA cycle. This shows that the difference in physiological states of lysine fermentation could be recognized at a protein level using 2-DE maps. Thus, the proteome analysis using 2-DE was found to be effective to obtain the intracellular information for the better understanding of bioprocesses.
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