Development of bioprocess for production of clean biodiesel fuel from waste oil

开发利用废油生产清洁生物柴油燃料的生物工艺

• 批准号: 13555208
• 负责人: KONDO Akihiko
• 金额: $ 8.38万
• 依托单位: Kobe University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13555208/
• 关键词: bioprocess; lipase; whole cell catalyst; yeast; biodiesel; methanolysis; methyl ester; cell surface display; 糸状菌; 環境調和型; メタノリシス反応; 細胞工場; 菌体触媒; 固定化; バイオリアクター

Biodiesel (Fatty acid methyl esters), which is derived from triglycerides by transesterification with methanol (methanolysis reaction), are renewable, biodegradable, and nontoxic fuel. Development of biodiesel fuel production process by using lipase without an organic solvent is very important. In this study, methanolysis of soybean oil by lipases from various microorganisms were investigated. Many lipases from various microorganisms were found to catalyze methanolysis in a water containing system without an organic solvent. Especially lipases from Rizopus oryzae, Candida rugosa, Pseudomonas cepacia, and Pseudomonas fluorescens showed high abilities to catalyze methanolysis. As a means of reducing the cost, the use of whole cell biocatalysts immobilized within biomass support particles (BSPs) was investigated. Rhisopus oryzae cells immobilized within BSPs and treated with glutaraldehyde was found to be very effective for biodiesel fuel production. In addition, yeast cells constructed by intracellular overproduction of Rhizopus oryzae lipase (ROL) are found to be effective for biodiesel fuel production. To further improve the activity of yeast cell, the new cell surface display systems based on the Flo1p flocculation functional domain was developed. Yeast cells displaying ROL and intracellularly expressing ROL showed the high methanolysis activity. The reactivity of ROL was improved by molecular engineering of ROL using a combinatorial protein library constructed on the yeast cell surface.

生物柴油（脂肪酸甲酯）是由甘油三酯与甲醇进行酯交换反应（甲醇分解反应）而得，是可再生、可生物降解且无毒的燃料。开发利用脂肪酶而不使用有机溶剂生产生物柴油燃料的工艺非常重要。在本研究中，研究了来自各种微生物的脂肪酶对大豆油的甲醇分解作用。发现来自各种微生物的许多脂肪酶在不含有机溶剂的含水系统中催化甲醇分解。特别是来自米根霉、皱褶假丝酵母、洋葱假单胞菌和荧光假单胞菌的脂肪酶显示出很高的催化甲醇分解的能力。作为降低成本的一种手段，对固定在生物质载体颗粒（BSP）内的全细胞生物催化剂的使用进行了研究。人们发现，固定在 BSP 内并用戊二醛处理的米根霉细胞对于生物柴油燃料的生产非常有效。此外，通过细胞内过量生产米根霉脂肪酶（ROL）构建的酵母细胞被发现可有效生产生物柴油燃料。为了进一步提高酵母细胞的活性，开发了基于Flo1p絮凝功能域的新型细胞表面展示系统。展示ROL和细胞内表达ROL的酵母细胞显示出高甲醇分解活性。使用在酵母细胞表面构建的组合蛋白库，通过 ROL 的分子工程提高了 ROL 的反应性。

项目成果

Takeshi Matsumoto: "Construction of yeast strains with high cell surface lipase activity by using novel display systems based on the Flo1p flocculation function domain"Applied and Environmental Microbiology. 68. 4517-4522 (2002)

Takeshi Matsumoto：“通过使用基于 Flo1p 絮凝功能域的新型展示系统构建具有高细胞表面脂肪酶活性的酵母菌株”应用和环境微生物学。

Kazuhiro Ban: "Repeated use of whole-cell biocatalysts immobilized within biomass support particles for biodiesel fuel production"Journal of Molecular Catalyses B. 17(3-5). 157-165 (2002)

Kazuhiro Ban：“重复使用固定在生物质载体颗粒内的全细胞生物催化剂用于生物柴油燃料生产”《分子催化杂志》B. 17(3-5)。

Masaru Kaieda: "Effect of methanol and water contents on production of biodiesel fuel from plant oil catalyzed by various lipases in a solvent-free system"Journal of Bioscience and Bioengineering. 91(1). 12-15 (2001)

Masaru Kaieda：“甲醇和水含量对无溶剂系统中各种脂肪酶催化的植物油生产生物柴油燃料的影响”生物科学与生物工程杂志。

Kazuhiro Ban: "Whole cell biocatalyst for biodiesel fuel production utilizing rhizopus oryzae cells immobilized within biomass support particles"Biochemical Engineering Journal. 8. 39-43 (2001)

Kazuhiro Ban：“利用固定在生物质支持颗粒内的米根霉细胞生产生物柴油燃料的全细胞生物催化剂”《生物化学工程杂志》。

Kazuhiro Ban: "Whole cell biocatalyst for biodiesel fuel production utilizing rhizopus oryzae cells immobilized within biomass support particles"Biochemical Engineering Journal. 8(1). 39-43 (2001)

Kazuhiro Ban：“利用固定在生物质支持颗粒内的米根霉细胞生产生物柴油燃料的全细胞生物催化剂”《生物化学工程杂志》。