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

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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.