Characterization And Catalytic Properties Of Candida Antarctica Lipase B-Displaying Pichia Pastoris Cells In Non-aqueous Phase

Candida antarctica lipase B (CALB) exhibit better catalytic activity than the otherlipases in the esterification, hydrolysis, transesterification, as well as other types of reactions.Analogous to conventional enzyme immobilization, the display of lipases can be consideredas a type of self-immobilization on cell wall by connected to a anchoring protein. Suchlipase-displaying yeast cells can be produced during a high density fermentation, and afterlyophilization, the cells can be directly uesd as biocatalyst without enzyme purification step.So this procedure prevents the enzyme loss during immobilization process and saves cost.However, little has been known about catalytic characteristics of displayed lipases innon-aqueous phase. In this work, the CALB-displaying Pichia pastoris whole-cells were usedto catalyze various reactions. Substrate selectivity, solvent tolerance and reaction kineticswere discussed to determine the catalytic characteristics of CALB-displaying P. pastoris cellsin non-aqueous phase. The relationship between cell surface characteristics and catalyticsynthesis efficiency was also investigated by cell surface modification. Specific contents andresults are as follows:(1) Quantificationof the enzyme displayed on cell surfaceLipase irreversible inhibitor4-methylumbelliferyl hexylphosphonate (4-MUHP), whichcan irreversiblely bind with the lipase activity site and release a fluorescent substance4-methylumbelliferyl (4-MU) is synthesized. By determination of4-MU amount, the number oflipase active site can be calculated. This method is accurate, sensitive and in good agreementwith hydrolysis activity and synthesis activity and is a powerful tool to evaluate thecharacteristics of CALB-displaying cells used as immobilized enzyme.(2) Synthesis of flavor esters catalyzed by CALB-displaying P. pastoris cellsCALB-displaying P. pastoris cells were used to catalyze synthesis of short-chain flavoresters in non-aqueous phase. The study of substrate selectivity indicated that hindrance ofbranched and large group was the most important factor. Effects of solvent, temperature,substrate concentration, substrate molar ratio and cells dosage on esterification wereinvestigated and the results showed CALB-displaying P. pastoris cells were good tolerancefor high substrates concentration, especially strong polar substrates. Based on these results, 10flavor esters were successfully synthesized in5L stirred reactor. Solvent-free synthesis ofethyl caproate was also achieved in a50L pilot reactor and the conversion rate reached90%.(3) Synthesis of biodiesel catalyzed by lipase-displaying P. pastoris cellsThe combined utilization of CALB and RML, separately displayed on P. pastoris cells,to produce biodiesel in co-solvent media was investigated. The synergistic effect of the twodisplayed lipases was to elimination of the rate-limiting step. The use of tert-butanol andisooctane as the co-solvent media was found to reduce the adsorption of glycerol onto cells.Scaled-up reactions using various types of feedstock were carried out in a0.5L stirred reactorunder optimum conditions, affording methyl ester yields over90%in12h. Moreover, themethyl ester yields remained above85%after20repeated batch cycles.(4) Regioselective synthesis and enantioselective resolution catalyzed by CALB-displaying cellsCALB-displaying P. pastoris cells were used to catalyze the synthesis of glucose laurateand fructose laurate (mono-ester and di-ester). NMR detection of sugar ester shoued thatCALB exhibited specific selectivity for primary hydroxyl groups. CALB-displayingP.pastoris showed excellent R-enantioselectivity to-phenylethanol. However, poor substrateand product enantiomer excess values were obtained when catalyzing enantioselectiveresolution of bambuterol.(5) Surface hydrophobicity modification of CALB-displaying P. pastoris cellsFungal hydrophobin proteins HFBI, surfactant Tween80, ionic liquid [Omin][PF6], andhydrophobic carbon source were used to modify surface hydrophobicity of CALB-displayingP. pastoris cells. The CALB loading and hydrophobicity of modified cells were detectied; thechange of cell surface groups was characterized using Fourier transform IR. It was found thatfour modification methods had enhanced hydrophobicity of cell surface, Tween80and [Omin][PF6] absorbed on cell surface showed better enhancement. The modified cells were used tosynthesis of ethyl caproate, long-chain fatty acid ethyl ester and biodiesel, the catalyticefficiency were improved, especially for Tween80and [Omin][PF6]–modified cells.In this work, various types of reactions catalyzed by CALB-displaying P. pastoris cellshave been investigated to illustrate the catalytic properties of this biocatalyst whichperformed as a special form of immobilized enzyme in non-aqueous phase. The method to improve the cell surface properties was development, this may provide a reference forenzyme-displaying cells’ potential applications.