| As a green and renewable energy source, biofuel, has attracted increasing attention. Biofuel production catalyzed by lipase has been developed, due to its advantages, such as mild reaction condition, non-pollution and low emission. Moreover, with improved immobilization methods of lipase, the application of lipase catalysts has been great promoted. Surface displaying technique for lipase has combined separation, purification and immobilization, as well as decreased the cost of lipase immobilization. In the study, the Aga2 was used as anchor protein to display Rhizopus oryzae lipase (ROL) on S. cerevisiae cell surface. We constructed the whole-cell catalyst, characterized its enzymatic properties and initially evaluated the ferment cost as well as application in biofuel.1. Using bio-informational analysis to determine the matching rules of surface displaying units, such as anchor protein, host cell and target lipase, laid the theoretical basis for whole-cell catalyst. In structural analysis of 4 kinds of lipases and anchor proteins, ROL was selected as an ideal target protein, owning uniform hydrophobicity and flexibility. Its catalytic triad is formed by S242, D301 and H354, and T213 and L267 make up oxyanion hole. The active site locates near its C-terminal. Additionally, we choose Aga2-Agal of a-agglutinin as an appropriate anchor protein.2. Successfully using Aga2-Agal of a-agglutinin as anchor protein to display ROL on the S. cerevisiae cell surface, we achieved the whole-cell catalyst, which hydrolysis activity of olive oil was 78.9U·g-1 dry cell, superior to the reported, and its stability and organic solvent tolerance has been improved.3. Initially evaluating the ferment cost as well as application in biofuel, we obtained more than 3g mycelium biomass in 2L fermentation. The ferment cost was 68.9 yuan·g-1 in rough calculation. The ME yield of immobilized recombinant S. cerevisiae by alginate sodium was 81.2%, and it could remain 80% yield after 7 batches. |
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