Surface Display Of Geotrichum Candidum Lipase In Saccharomyces Cerevisiae And Pichia Pastoris

Lipases (EC3.1.1.3) have been widely used as important biocatalysts in industrialapplications, owing to their unique substrate selectivity, mild operation conditions andnarrow control of product distribution. However, the high cost and relative low operationstability of free enzyme are the key factors that have restricted the industrialization oflipase-catalyzed reaction. In this study, lipase from Geotrichum candidum was displayedon the cell wall of Saccharomyces cerevisiae and Pichia pastoris for the first time,yielding whole cell biocatalysts of lower cost and higher stablity. Furthermore, thecharacterizations of the displayed GCL including optimum temperature, optimum pH andthermo-stability were investigated. Most important, the GCL-displaying P. pastoris cellswere successfully applied as whole-cell biocatalysts into hydrolysis of fish oil forenriching EPA and DHA. The main contents and inovations of this study weresummarized as follows:1. Geotrichum candidum lipase was displayed on the cell wall of S. cerevisiaeEBY100using a-agglutinin as anchor protein for the first time. The activity of displayedGCL reached its maximum value43.7±0.5U/g dry cell after induction at20°C for72h,with an optimal pH and temperature at8.5and40°C. Besides, the display GCL exhibitedhigher thermo-stability than the free one, and retained85%of the original activity afterincubation at40°C for3h.2. A novel system based on a-agglutinin from S. cerevisiae was developed forimmobilization of proteins in P. pastoris. Besides, GCL was displayed on the cell wall of P.pastoris X-33for the first time. After electroporation, the recombinants were subjected toplate screening and shaking flask fermentation. Then the recombinant strainX-33/pPICZXFa-△GCL5#exhibited the highest activity was obtained as a result.Subsequently, the medium and fermentation of the recombinant was optimized by flaskfermentation. The optimum conditions were: methanol1%(v/v), inoculum4%(v/v),induced initial pH7.0. The activity of displayed GCL reached273±2.4U/g dry celltowards olive oil after96h of cultivation, while that of original conditions was180±4.45U/g dry cell. 3. The GCL displayed on P. pastoris exhibited relative high stablility in the pH valuesfrom pH6.0to pH8.0and retained80%of their origin activity after incubation at45°Cfor4h, with an optimal pH and temperature at7.5,45°C using olive oil as a substrate. Bycontrast, the optimal pH and temperature of displayed GCL was9.0,40°C and retained80%of their original activity after incubation at40°C for6h towards pNPC.4. The GCL-displaying P. pastoris cells were first used as whole cell biocatalysts toenrich EPA and DNA from fish oil. The hydrolysis degree reached its maximum value at28.6%after16h of treatment. As a result, the contents of EPA reached1.85%and DHA30.86%, increased by1.21-fold and1.29-fold respectively. The total yield of EPA andDHA reached46.62%.