Production of recombinant nitrate reductase in Pichia

毕赤酵母重组硝酸还原酶的生产

• 批准号: 6519838
• 负责人: Ellen Ruth Campbell
• 金额: $ 28.49万
• 依托单位: NITRATE ELIMINATION COMPANY, INC.
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6519838
• 关键词: Arabidopsis; bioreactors; biosensor device; biotechnology; catalyst; corn; environmental toxicology; enzyme activity; fermentation; gene expression; immobilized enzymes; immunoaffinity chromatography; microorganism mass culture; microorganism metabolism; nitrate reductases; nitrates; plant extracts; protein engineering; protein purification; recombinant proteins; technology /technique development; water sampling /testing; water treatment; yeasts

DESCRIPTION (provided by applicant): Clean, safe water is the base of good public health. NECi develops and commercializes enzyme-based products for water testing and remediation. Currently, NECi is focused on the application of higher plant Nitrate Reductase (NaR) to problems caused by excess nitrate in the environment, a pollution and human health issue worldwide. In Phase I, we demonstrated that recombinant, catalytically active NaR from Arabidopsis thaliana (AtNaR) can be produced economically in the yeast Pichia pastoris, to make NaR-based products more cost-competitive. AtNaR was purified using affinity chromatography, then performance tested in NECi nitrate test kit products. We expressed a second recombinant NaR form, YNR 1, from the yeast Pichia angusta, a key step to increase expression of fully active NaR enzyme. Efforts to engineer the NaR gene were begun using YNRI, and we developed a unique down-sized" form with nitrate-reducing ability intact. In Phase II, we propose to continue to optimize corn and yeast NaR cDNA clones for improved expression and stability, and for efficient purification. Fermentation parameters and optimal gene construction for maximal NaR production will be investigated. We will further develop and characterize down-sized forms of NaR for testing and use in NECi 's electrically-driven immobilized enzyme biosensor and water treatment prototypes. PROPOSED COMMERCIAL APPLICATION: Not Available

描述（由申请人提供）：清洁、安全的水是良好的基础 公共卫生。 NECi 开发酶基水产品并将其商业化 测试和修复。目前，NECi 的重点是应用 高等植物硝酸盐还原酶（NaR）可解决因过量硝酸盐引起的问题 环境、污染和全球人类健康问题。在第一阶段，我们 证明来自拟南芥的重组、具有催化活性的 NaR 拟南芥 (AtNaR) 可以在毕赤酵母中经济地生产， 使基于NaR的产品更具成本竞争力。 AtNaR 纯化使用 亲和层析，然后在 NECi 硝酸盐测试套件中测试性能 产品。我们从酵母中表达了第二种重组 NaR 形式 YNR 1 安古斯塔毕赤酵母 (Pichia angusta)，增加完全活性 NaR 酶表达的关键步骤。 利用 YNRI 开始了对 NaR 基因的改造，我们开发了一种 独特的“缩小尺寸”形式，具有完整的硝酸盐还原能力。在第二阶段，我们 建议继续优化玉米和酵母 NaR cDNA 克隆以提高 表达和稳定性，以及高效纯化。发酵 最大 NaR 产量的参数和最佳基因构建将是 调查了。我们将进一步开发和表征缩小尺寸的 NaR 用于测试和用于 NECi 的电动固定化酶生物传感器 和水处理原型。 拟议的商业应用： 无法使用

项目成果

Structural basis of eukaryotic nitrate reduction: crystal structures of the nitrate reductase active site.

真核硝酸盐还原的结构基础：硝酸盐还原酶活性位点的晶体结构。

• 发表时间: 2005-04
• 作者: Fischer, Katrin;Barbier, Guillaume G;Hecht, Hans;Mendel, Ralf R;Campbell, Wilbur H;Schwarz, Guenter
• 通讯作者: Schwarz, Guenter

Viscosity effects on eukaryotic nitrate reductase activity.

粘度对真核硝酸还原酶活性的影响。

• 发表时间: 2005-07-15
• 作者: Barbier, Guillaume G;Campbell, Wilbur H
• 通讯作者: Campbell, Wilbur H

Purification and biochemical characterization of simplified eukaryotic nitrate reductase expressed in Pichia pastoris.

巴斯德毕赤酵母中表达的简化真核硝酸还原酶的纯化和生化表征。

• 发表时间: 2004-09
• 影响因子: 1.6
• 作者: Barbier, Guillaume G;Joshi, Rama C;Campbell, Ellen R;Campbell, Wilbur H
• 通讯作者: Campbell, Wilbur H