| With the gradual depletion of fossil oil, biodiesel, a renewable, non-toxic and biodegradable fuel, has gained more attentions in recent years for its ability to replace fossil fuels. The use of lipase instead of acid or alkaline as catalyst for biodiesel production has great market potentiality due to its environment-friendly property and mild reaction conditions. However, lipases have some disadvantages, such as high price and easily being inactivated in organic solvent, which restrict industrialization of this technology. The above problem could be solved by surface display of lipase, because surface display of lipases as a whole-cell catalyst can be recycled and the stability of lipase can be greatly improved in comparison with traditional immobilization of lipases. Compared with conventional immobilized enzymes, the whole-cell catalysts have unique advantages, such as a simpler product purification and a more cost-effective downstream processing. Furthermore, because the lipases are immobilized on the surface of cells, the substrates can be simply access to the enzyme and the products can be easily released. Additionally, surface display of lipase can also serve as an efficient high-throughput screening method for protein engineering of lipase. The main results of this study were summarized as follows:1. Through bioinformatic analysis, we have learnt many characteristics of Yarrowia lipolytica lipase Lip2, such as hydrophobicity, flexibility, secendary structure and tertiary structure. Its catalytic triad consists of S162, D230 and H289. T88 and L163 form the oxyanion hole. Its activity centre is close to the C-terminal.2. Lip2 from Y. lipolytica was displayed on the cell surface of Saccharomyces cerevisiae using Cwp2 as an anchor protein for the first time. Successful display of the lipase on the cell surface was confirmed by immunofluorescence microscopy and halo assay. The length of linker sequences was further examined to confirm whether the correct conformation of Lip2 was maintained. The results showed that the displayed Lip2 exhibited the highest activity at 7.6 U/g-dry cell when using (G4S)3 sequence as the linker, with an optimal temperature and pH at 40℃and pH 8.0. The displayed lipase did not lose any activity after being treated with 0.1% Triton X-100 and 0.1% Tween 80 for 30 min, and it retained 92% of its original activity after incubation in 10% DMSO for 30 min. It also exhibited better thermostability than free-form Lip2 as reported previously.3. A Flol fragment consisting of 469 amino acid residues was used as an anchor protein for surface display of Lip2 in S. cerevisiae. Successful surface display of Lip2 was also confirmed by immunofluorescence microscopy and halo assay. The results showed that the highest activity of displayed Lip2 was 65.2 U/g-dry cell, much higher than that of using Cwp2 as anchor protein. Compared with the displayed Lip2 using Cwp2 as an anchor protein, the stability against SDS and DMSO was slightly decreased. The optimal temperature and pH, as well as the thermostability were consistent with that of using Cwp2 as anchor protein.4. It is also for the first time that the a-agglutinin was used as an anchor protein for surface display of Lip2. From immunofluorescence microscopy and halo assay was successful surface display of Lip2 in S. cerevisiae confirmed. The activity of displayed Lip2 reached 634.9 U/g-dry cell, much higher than those of using Cwp2 and Flol fragment as anchor proteins. The optimal temperature and pH was 40℃and pH 8.0. Compared with the displayed Lip2 using Cwp2 and Flol fragment as anchor proteins, the thermostability and the stability against organic solvent were much improved. Especially, the displayed Lip2 remained 109.4% and 98.5% of its original acitivity after being treated with 20% methanol and ethanol, much better than free-form Lip2, which suggests that the displayed Lip2 will be more suitable for biodiesel production.5. By comparing the results of surface display of Lip2 using three different anchor proteins, we have preliminarily learned the influence of different anchor proteins on the conformation of displayed Lip2. Based on this, we bring forward a rough principle on how to select a suitable anchor protein for surface display of a target lipase. It says that the structure feature of the target protein should first be assayed through bioinformatics and then the cell wall protein with its anchor domain far away from the activity centre of the target protein should be chosen as the anchor protein. By following this principle, we successfully displayed Y. lipolytica lipases Lip7, Lip8 and Aspergillus niger lipase (ANL) on the surface of S. cerevisiae. The displayed Lip7 and Lip8 showed high activities, which reached 282.9 U/g-dry cell and 121.3 U/g-dry cell, respectively. The thermostability of displayed Lip7 and Lip8 were much better than that of the displayed Lip2. Each displayed lipase retained above 90% of its original activity after incubation at 50℃for 5 h. However, the surface displayed ANL exhibited a relatively low activity of 43.8 U/g-dry cell. It could also remain more than 95% of its original activity after incubation 50℃for 4 h.
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