成果報告書詳細
管理番号100013787
タイトル*平成20年度中間年報 「微生物機能を活用した環境調和型製造基盤技術開発/微生物機能を活用した高度製造基盤技術開発 高性能宿主細胞創製技術の開発、微生物反応の多様化・高機能化技術の開発(発現タンパク質解析による微生物機能利用のための技術基盤の研究開発)」(H18-H21)
公開日2009/4/24
報告書年度2008 - 2008
委託先名独立行政法人製品評価技術基盤機構
プロジェクト番号P06014
部署名バイオテクノロジー・医療技術開発部
和文要約以下本編抜粋:近年、資源枯渇やCO2等排出物の環境への影響が懸念されており、循環・環境調和型産業のため製造技術基盤として、微生物機能を活用した物質生産(以下、「バイオプロセス」という。)を強化・発展させる必要性が出てきた。
英文要約Title: Environmentally-friendly fundamental processing technology utilizing microbial functions / Development of basic technologies for advanced production methods using microorganism functions / High-performance host cell creation technology, Technology to enable enhanced and diversified functions of microbial processes (Development of the technology base by the protein expression analysis for utilizing microbial functions) (FY2006-FY2009) FY2008 Annual Report (1) Purpose of this project There are increasing concerns about environmental impacts of the exhaustion of natural resources and the emission of CO2. To establish technological basis for environmentally-friendly and sustainable industries, we need to develop advanced methods for material production utilizing microbial functions (bioprocess). Consequently, it is of primary importance to develop the technology for creating high-performance host cells, and the technology to enable enhanced and diversified functions of microbial processes. In fiscal 2008, Department of Biotechnology, National Institute of Technology and Evaluation (NITE-DOB) planned to perform the comprehensive protein expression analysis of Rhodococcus opacus strain B4 (R. opacus B4), chosen as an organic solvent-resistant microorganism, to contribute to the development of the technology to enable enhanced and diversified functions of microbial processes. (2) Study on culture conditions for R. opacus B4 R. opacus B4 was cultured either in aqueous medium or biphasic aqueous/organic medium and its proteome was analyzed comprehensively by the following methods. (3) Comprehensive proteome analysis by shotgun method Proteins were extracted from the cells and separated by one-dimensional gel electrophoresis (SDS-PAGE). Separated proteins were digested and resultant peptides were analyzed by the high-performance liquid chromatography - electrospray ionization - tandem mass spectrometry (LC-ESI-MS/MS). (4) Comprehensive proteome analysis by two-dimensional gel electrophoresis Proteins extracted from the cells were separated by two-dimensional gel electrophoresis. Each protein was digested and subjected to MALDI-TOF mass spectrometry for protein identification. (5) Analysis of differentially expressed proteins by comprehensive proteome analysis Two stock cultures of R. opacus B4 (B4N and B4H) with different levels of organic solvent resistance were used for proteome analysis. Proteins identified under two culture conditions were compared and a list of differentially expressed proteins was made to assist further studies for the elucidation of molecular mechanisms of the organic solvent resistance. Proteins whose expression levels were different between two stock cultures were categorized according to their predicted functions. As a result, proteins related to metabolism were more abundantly expressed in B4H than in B4N. Changes in expression levels are being analyzed in further detail for each of the metabolic pathway.
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