High Cell-density Fermentation Of Geotrichum Candidum Lipase GCL In Recombinant Pichia Pastoris

The Geotrichum candidum lipase (GCL) is a catalyst with substrate specificity to thefatty acids owning a cis-double bond in the9-position. Thus, GCL can be used toselectively hydrolyze fatty acids to enrich unsaturated fatty acids (UFAs) with highnutritional value or to selectively esterificate and separate conjugated linoleic acid isomers.The objectives of this thesis are to optimize shake flask fermentation conditions andhigh-density fermentation conditions of the recombined Pichia pastoris harboring GCL Bgene and appearing Mut+phenotype and Mutsphenotype, for enzyme production in10-Lfermenter. The main results are as follows:(1) Rhodamine B flat and shake flask fermentation were employed to quanlitativelyand quantitatively detect the enzyme activity of X-33/pPICZα-gcl57#. The initial enzymeactivity is140U/mL (olive oil emulsion, alkali type titration). The optimized shake flaskfermentation conditions were40mL culture medium in500mL shake flask, pH7.0,1%methanol, and induced72hours with the highest enzyme activity at375U/mL. Then, thefermentation conditions of X-33/pPICZα-gcl57#in10-L fermenter were optimized, andthe optimal conditions of enzyme production were: fermentation temperature29.0℃, pH5.0in cell growth phase, and the temperature27.0℃, pH6.0in the induction phase. Undersuch conditions, the biomass reached338g/L, the protein content2.68g/L, the enzymeactivity5,000U/mL, and the productivity of enzyme activity30.69U/(mL h) after162.8hours. With methanol and glycerol mixed fed culture, the biomass attained at398g/L, theprotein content at2.56g/L, enzyme activity at5,600U/mL, and the productivity ofactivity enzyme at40.06U/(mL h). Compare with methanol fed culture, the enzymeactivity increased by25.0%, and the productivity of enzyme activity by78.2%.(2) The pPICZα-gcl vector was imported into the P. pastoris KM71H by electricalconversion, and the recombinant33#with muts phenotype was sieved. The highestenzyme activity was up to215U/mL, and the optimal loading dosage of methanol forenzyme production was0.6%. The biomass reached332g/L, the protein content2.61g/L,the enzyme activity5,450U/mL, and the productivity of enzyme activity27.69U/(mL h) after196.77hours under high-density fermentation in10-L fermenter. While the biomassreached394g/L, the protein content3.06g/L, the enzyme activity6,500U/mL, and theproductivity of enzyme activity38.62U/(mL h) after168.3hours with methanol andsorbitol mixed fed culture.(3) The mixed carbon source fed fermentation was explored with the dual-promoterrecombinant, GS115/9Kgcl-FLDgcl26#in10-L fermenter. The biomass reached396g/L,the protein content4.52g/L, enzyme activity11,200U/mL, and the productivity ofactivity enzyme98.98U/(mL h) after113.1hours with methanol and sorbitol mixed fedculture.(4) The following conclusions are drawn by comparing the fermentationcharacteristics and enzyme production of the three engineering strains in the10-Lfermenter: i. Changes in parameters of Mutsare more stable than those of mut+; ii.methanol and sorbitol mixed fed culture can significantly reduce the production ofpigment (FAD); iii. the enzyme activity increased20%and the productivity of enzymeactivity increased by30%-60%with methanol and sorbitol mixed fed culture incomparison to methanol fed culture alone; iv. the enzyme activity increased by ca.100%and the productivity of enzyme activity also increased by100%with the dual-promoterrecombinants by genetic engineering in comparison to the single-promoter recombinants.