High-Level Expression Of Thermomyces Lanuginosus Lipase In Pichia Pastoris And Its Application In Biodiesel Preparation

With the shortage of petroleum and the reemergence of the consciousness for environmental protection,biodiesel fuel(fatty acid methyl esters,FAMEs)produced by transesterification between plant oil and short chain alcohol was expected as one of the most important substitutes for fossil diesel fuel.So far,the commercial biodiesel has been produced mainly by chemical methods,to which excessive energy consuming and environment pollution are its major drawbacks.In non-aqueous medium,lipases(EC 3.1.1.3)exhibited novel transesterification properties that make thempromising biocatalysts forbiodieselpreparation.Comparing with chemical methods,enzymatic methods has great advantages as reduced energy consume,simplified the recovery of FAMEs,and eliminated the environment pollution.However,the production of biodiesel fuel by lipase has not yet been applied industrially.To make the enzymatic method economically more competitivethan the chemical method,the development of stable,active lipase,and realized its high-level productionis crucial for its industrial application.Lipase TLL from the thermophilic fungus Thermomyces lanuginosus was high stable to temperature,tolerant to organic solvent and efficient for transesterification reaction in non-aqueous medium.It was regarded as an ideal biocatalysis for biodiesel preparation.In order to make the lipases reusable and improve their economical competence in industrial application.In this project,we have successfully realized the high-ratio conversion of plant oil into FAMEs by directly using the liquid enzyme from the culture of lipase recombinants.We firstly achieved the high-level secretory expression of Thermomyceslanuginosus lipase by methods of in silica gene design and codon optimization,increasingthe gene dosages in genome,the enzyme activity reached 432 U/mL and the protein content reached 1.58 mg/mL.Through 14 L fermentation tank fermentation,the enzyme activity reached 875 U/mL,and the protein content reached 3.22 mg/mL,then directly used the liquid lipase as the catalyzer for FAMEs production by transesterification between plant oil and methanol.After reaction parameters optimization,about 96% conversion ratiowas achieved.The strategy by directly using the liquid culture of lipase recombinantsas the biocatalyer for FAMEs preparationhas greatly simplified the procedures of enzyme immobilization and rejuvenation,FAMEs recovery,and present an alter native of the enzyme forms in non-aqueous catalysis systems.