Surface Display Of Candida Antarctica Lipase B And Thermomyces Lanuginosus Lipase With Synergy In Pichia Pastoris

The cell surface display technique can make foreign protein be fused with the surfaceof microorganisms and retaining its bioactivity. This technique is now broadly used inwhole cell catalyst, antibody production, biosensor, vaccine, and so on. Both Candidaantarctica lipase B and Thermomyces lanuginosus lipase are lipases with particularselectivity, being applied in wide industry fields. However, so far, there is no report onco-displayed lipases on yeasts. Therefore, we displayed these two lipases on the cellsurface of Phichia pastoris to construct a novel whole-cell catalyst with greater enzymeactivity and catalyst efficiency. It is regarded as a significant work, and will provide asolidated technology for their commercializing usage.Firstly, we evaluated display effects of several anchor proteins fused with fluorescentprotein EGFP or RFP in pPIC9k and pPICZαA with the analysis results of crystalstructures of the lipases, respectively. Then a proper system was chosen to make aconstruction of single display system of CALB or TLL, respectively. Consequently, aco-display system containing both CALB and TLL was constructed by secondelectro-transformation. The main results of this study were summarized as follows:1. A series of expression systems based on the pPIC9k harboring target protein EGFPfused with anchor proteins of Cwp2p, Sed1p and α-agglutinin were obtained. Afterfermentation, the recombinant cells were evaluated by displaying rate with flow cytometer.The result was that Cwp2p, Sed1p and α-agglutinin respectively fused with EGFP showeddisplaying rates53.35%,99.5%and46.26%. The anchor protein Sed1p was the best andwas chosen to fuse with lipase CALB in pPIC9k system. A display vector pPIC9k-CASwas constructed and then transformed to GS115. The recombinants were screened andfinally, a recombinant with the activity of85.5U/g dry cells was obtained. Thefermentation cells under the fluorescence microscope emitted bright green fluorescenceand the displaying rate detected was98.81%. The enzymatic properties of the displayedCALB were studied, and the results are: the optimum temperature is40℃and the pH is8.5; after1h incubation at40℃and60℃, the activity retained85%and30%,respectively. The irons K²⁺, Ca²⁺, Mg²⁺caused weak activation to the displayed CALB; Mn²⁺, Cu²⁺, EDTA, DMSO, TritonX100and methanol showed inhibition effect on thewhole-cell catalyst, and SDS showed little impact.2. Two expression systems based on the pPICZαA containing target protein RFP fusedwith anchor proteins of Flo1p and Sed1p were obtained. After fermentation, therecombinant cells were evaluated according to the displaying rate with flow cytometer.The Flo1p and Sed1p respectively fused with RFP showed displaying rates24.66%and98.36%. A display vector pPICZαA-TLS was constructed on the base of Sed1p and thentransformed to GS115. At last, a recombinant with the activity of257.8U/g dry cells wasobtained. The treated fermentation cells under the fluorescence microscope emitted brightred fluorescence, and the displaying rate was detected98.02%. The enzymatic propertiesof the displayed TLL were studied, the optimum temperature is30℃and the pH is8.0;after1h incubation at40℃, the activity retained95%. The irons K²⁺, Ca²⁺, Mg²⁺exhibited weak activation to the displayed TLL; Mn²⁺, Ni2+, EDTA, SDS and Tween20showed little inhibition effect to the whole-cell catalyst, and Cu²⁺showed a strongdepression effect.3. The display vector pPICZαA-TLS was then transformed into GS115/pPIC9k-CAS. Therecombinants were screened and a recombinant containing two lipases with the activity of623U/g dry cells was obtained, which is7.3and2.4times as the single displayed CALBand TLL. The treated cells containing two lipases under the fluorescence microscopeemitted bright red fluorescence and green fluorescence at the same time, and thedisplaying rates were94.86%and74.45%detected by Flow Cytometer. The displayedCALB and TLL showed excellent thermostability and good tolerance to organic solventstolerance, with an optimal temperature and pH at30℃and8.5. K²⁺, Ca²⁺, Mg²⁺, EDTA,DMSO, TritonX-100, SDS caused weak activation to the whole-cell catalyst, while Cu²⁺and some short chain alcohols showed strong inhibition effect. As a whole-cell catalyst,the fermentation cells co-displayed two lipases were used to prepare biodiesel fromsoybean oil, and the biodiesel yield was72.62%, which is11.04%and12.62%more thanthe single displayed CALB and TLL, respectively.