Surface Display Of Aspergillus Niger Lipase A In Pichia Pastoris

Aspergillus niger is considered to be GRAS (Generally regarded as safe) by theUnited States Food and Drug Administration (FDA). Lipases from A. niger possessenantioselectivity and positional selectivity toward the sn-1,3positions of the triglyceridemolecules. Therefore, they are widely used in industries, such as food additives, oil-fatmodification, enantiomer resolution and detergent additives, etc. However, free A. nigerlipases can be easily deactivated in organic solvent and cannot be recycled, whichobstructs their application in industry. To slove the above problems of free lipase, aconcept of self-immobilized whole-cell catalyst was applied here, and A. niger lipase A(ANLA) was displayed on the yeast cell surface by cell surface engineering. The displayedlipase can be reused easily and more stable than free form. However, the activity of thedisplayed lipase is influenced by many factors. In early research of our laboratory, the cellwall protein Sed1from Saccharomyces cerevisiae was screened with sound displaying rate.In this study, Pichia pastoris X-33was chosen as the host to display ANLA owing to itsless hypermannosylation than S. cerevisiae, with Sed1as the auchor protein. The effects ofdifferent linker sequences and promoters on the activity of the displayed ANLA wereinvestigated. The aim of this study is to provide theoretical basis and technical support toprepare a whole-cell biocatalyst with better properties of A. niger lipase. The main resultswere summarized as follows:1. The C terminus of ANLA was fused to the Sed1anchor protein and the resultingfusion protein was expressed on the cell surface of P. pastoris X-33. Linker peptidesconsisting of Gly/Ser or Ser/Thr repeated sequences were inserted between ANLA andSed1. Six high lipase activities of recombinants possessing different linker sequencenamed X-33/pAOX-A0S89#, X-33/pAOX-AGS44#, X-33/pAOX-AS1S44#,X-33/pAOX-AS2S32#, X-33/pAOX-AS3S42#, X-33/pAOX-AS4S62#were screened.The lipase activities of these recombinants were multiply compared by Fisher method (α=0.05). The lipase activities of X-33/pAOX-AGS44#(24.96U/g dry cell) andX-33/pAOX-AS1S44#(22.74U/g dry cell) had no significant difference (p>0.05).However, they were significantly higher than those of the other four recombinants. The recombinant cells were evaluated by displaying rate with flow cytometer. The displayingrate of X-33/pAOX-AGS44#and X-33/pAOX-AS1S44#were the highest (94.58%) andthe lowest (61.57%), respectively. The above results showed that the displayed ANLAexhibited the highest activity and displaying rate when using (G4S)3as linker sequence.The optimal temperature and pH of the displayed ANLA was45°C and pH7.5. Comparedwith free ANLA, the stability of the displayed ANLA was improved to some extent.2. The inductive expressing vector pFLD-AGS and constitutive expression vectorspGAP-AGS, pTEF-AGS were constructed. Three recombinants of X-33/pFLD-AGS35#,X-33/pGAP-AGS19#, X-33/pTEF-AGS77#with high activities were screened. Theactivities of these recombinants and X-33/pAOX-AGS44#were multiply compared.X-33/pAOX-AGS44#and X-33/pFLD-AGS35#had no marked difference (p>0.05).However, they were significantly higher than those of the other two recombinants. Theactivity of X-33/pTEF-AGS77#was distinctly less than those of the other threerecombinants.3. The culture conditions of recombinants X-33/pAOX-AGS44#, X-33/pFLD-AGS35#, X-33/pGAP-AGS19#were optimized by flask fermentation, respectively. Theoptimized conditions were inoculum6%, loading1%methanol into500mL shaking flaskcontaining50mL medium each day, lasting72h at pH6.0, under which the lipase activityof recombinant X-33/pAOX-AGS44#reached38.02U/g dry cell. While the optimalconditions of recombinant X-33/pFLD-AGS35#were methanol induction72h, initial pH7.5, adding2%methanol per24h, inoculum2%, and40mL medium. The maximumlipase activity attained30.46U/g dry cell. For X-33/pGAP-AGS19#, the optimal cultureconditions were oil acid as carbon source, peptone4%, yeast extract1%, initial pH6.5,inculum2%, and50mL medium, its maximum lipase activity reached27.06U/g dry cellafter48h of cultivation. Then, the maximum lipase activiities of the above threerecombinants were multiply compared. The activity of X-33/pAOX-AGS44#wassignificantly higher than those of the other two. The above results indicated that thehighest lipase activity was obtained with X-33/pAOX-AGS44#which expresseddisplayed ANLA by the AOX1promoter.