Influence Of Displayed Fungal Hydrophobin To Cell-surface Hydrophobicity Of Pichia Pastoris And To Co-displayed Candida Antarctica Lipase B

The yeast express system is a kind of rapidly growing and extensive applied eukaryoticcell expression system. The Pichia surface display system based on Pichia cell aim to expresstarget proteins on the host cell surface by an anchor of cell wall protein. It is a usefultechnique for protein related researches since Pichia cell is easy to cultivate in a low-costmedium and the target proteins can be expressed efficiently, as well as the Pichia cell is ableto be recovered and reused conveniently. In order to increase the surface hydrophobicity ofPichia pastoris cell, making the recombinant Pichia cell a more appropriate vector fordisplayed enzyme catalyzing, the fungal hydrophobins, HFBI and SC3, were displayed onPichia cell surface respectively. The influence of the hydrophobin to host surfacehydrophobicity and to the characteristic of Candida antarctica lipase B （CALB） co-displayedon the host cell were estimated respectively. The specific research content is list below.（1） Displaying hydrophobin on the surface of P. pastorisA HFBI surface-displayed recombinant Pichia strain ZfH21and a SC3surface-displayed recombinant Pichia strain ZfS21were constructed in this part. The“CALB-hydrophobin” fusions were identified to be covalently immobilized on the host cellwall by glycophosphatidylinositol （GPI） anchor in Western Blot assay. And theimmuno-fluorescence analysis was performed to confirm that the fusions were really stayedon the host cell wall. Then, the relative amount of fusions was estimated by counting thefluorescence intensity of ten thousand cells of each sample. Results indicated that more than52percent of hydrophobin was expressed on ZfH21strain surface compared with ZfS21strain. In addition, compared with the control strain Z21that does not have hydrophobindisplayed on its surface, the cell surface hydrophobicity of ZfH21strain was8.0fold high asZ21strain, and the cell surface hydrophobicity of ZfS21strain was6.3fold high as Z21strain.This result indicated that hydrophobins were functional displayed on the host cell surface.（2） Co-displaying hydrophobin with CALB on the surface of P. pastorisIt is the first time to co-display hydrophobin and enzyme on the surface of P. pastoris aswe know. A CALB displayed Pichia strain KhCSa was constructed firstly using aSaccharomyces cerevisiae cell wall protein Sag1p C-terminal as anchor. The hydrolytic activity （623U/g-dry cell） and synthetic activity （277U/g-dry cell） of KhCSa strain weredetected. The CALB/hydrophobin co-displayed Pichia strains, KhCSa:ZfH21andKhCSa:ZfS21, were constructed based on a KhCSa strain. The cell surface enzymatic activity,the amount of CALB, and the hydrophobicity of each strain were assayed. Results showedthat KhCSa:ZfH21and KhCSa:ZfS21strains possessed151.2%and38.5%of enzymaticactivity compared with KhCSa strain respectively. They possessed, however,90.1%and12.1%of CALB amount compared with KhCSa strain respectively. It is illustrated that hydrophobincan promote the CALB activity when they were displaying together on a Pichia cell surface.No matter displayed on KhCSa strain surface or on the co-displayed strains surface, theCALB lipases possessed similar optimum pH, optimum temperature, and optimum substrate.The CALB In addition, both KhCSa:ZfH21and KhCSa:ZfS21strains have lowerhydrophobicity,96.1%and42.8%, compared with KhCSa strain. It is indicated that the cellsurface hydrophobicity is not only affected by hydrophobin, but also affected by theco-displayed CALB.（3） Construction of a co-display system based on a FMDV2A peptideIt is the first time to bring the foot-and-mouth disease virus （FMDV）2A peptide inconstruction of a co-display system in P. pastoris as we know. A CALB displayed Pichiastrain KfCSe was constructed firstly using a Saccharomyces cerevisiae cell wall proteinSed1p as anchor. The hydrolytic activity （529U/g-dry cell） and synthetic activity （217U/g-dry cell） of KfCSe1strain were detected. The double CALB co-displayed Pichia strainsKfCSe2AhCSa was constructed based on a KhCSa strain. It used a2A peptide to mediateco-display of upstream protein CALB-Sed1p and downstream protein CALB-Sag1p. OnKfCSe2AhCSa strain surface, the protein amount of CALB-Sed1p was equal to94.3%ofKfCSe1strain, and the CALB-Sag1p amount was equal to63.9%of KhCSa strain. It isindicated that2A peptide seriously blocked the expression of downstream protein in Pichiasurface-display system, but almost didn’t affect the downstream protein. Interestingly, thehydrolytic activity of KfCSe2AhCSa strain was nearly equal to the proportional sum ofactivity of KfCSe1and KhCSa, which means the enzymatic activity of KfCSe2AhCSa strainwas proportional to the CALB displayed on its surface. Furthermore, two Pichia strain,KfH212AhCSa and KfS212AhCSa, were constructed using2A peptide to mediate hydrophobin/CALB co-display. The hydrolytic activity of KfH212AhCSa strain （427U/g-drycell） and KfS212AhCSa strain （70U/g-dry cell） were measured, which meams that2Apeptide was able to mediate co-display of hydrophobin and CALB in a Pichia host cell.（4） Preliminary study of directed CALB immobilization based on hydrophobinA CALB-HFBI fusion expressed recombinant Pichia strain KfCH and a CALB-SC3fusion expressed recombinant Pichia strain KfCS were constructed in this part. The hydrolyticactivity and synthetic activity of KfCH strain （329U/g-dry cell,203U/g-dry cell） and KfCSstrain （92U/g-dry cell,89U/g-dry cell） were detected respectively. The estimatedconcentrations of fusion in broth were5.6mg/mL CALB-HFBI for KfCH and0.4mg/mLCALB-SC3for KfCS by SDS-PAGE assay. The KfCH strain was remained31%hydrolyticactivity after kept in55oC envelopment for2h. A hydrophilic material SiO₂powder and ahydrophobic material centrifuge tube （polypropylene） were used to assess the effect ofdirected immobilization of “CALB-hydrophobin” fusion. Result showed that CALB-SC3immobilized on the surface of SiO₂and centrifuge tube inner surface more firm thanCALB-HFBI. The amount of CALB-SC3required for completely immobilization onpolypropylene surface was estimated to be0.82μg for one square meters.