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Expression Optimizing Strategy Of Recombinant Human Lysozyme And The Application Of Infrared Spectrometer On The Substrate Monitor In Pichia Pastoris Fermentation

Posted on:2015-01-14Degree:MasterType:Thesis
Country:ChinaCandidate:S WuFull Text:PDF
GTID:2251330428978065Subject:Biochemical Engineering
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Human Lysozyme (HLZ) is a kind of medicines and healthy product that has a broad market prospect. This thesis reported the research on the physiological characteristics of the expression of recombinant human lysozyme by Pichia pastoris fermentation and the application of infrared spectrometer on Pichia pastoris fermentation has carried on a preliminary study.The culture conditions was optimized by the response surface analysis methods in shake flasks, enzyme activity reached the4210U/mL compared that before optimization. The effect of osmotic stress on the production of Human Lysozyme by Pichia pastoris was quantitatively studied. Osmolarity progressively increased by addtion of NaCl in the culture broth, leading to decrease in cell growth, but the protein productivity was enhanced when introducing O.lmol/LNaCl, Yp/X could be increased notably from342.42to383.36U/g which is demonstrated an optimal producing osmolality depending on the strain. Cytomembrane fatty acids contents were also changed dramatically to enhancing the membrane fluidity to adapt to osmotic stress. Proline was studied in order to decrease the inhibitory effects of cell growth by osmotic stress to improve the production. When1.5g/L of proline as a compatible solute was added to the culture medium, it was imported and enhanced cell growth and the HLZ activity about10.9%and17.4%in the flask culture and chemostat culture respectively.Three supplementary carbon sources, glycerol, sorbitol and lactic acid was added in the induction phase to investigated the influence on growth and expression, it found that methanol feeding with sorbitol has an obviously positive effect on the production of HLZ. By adding the sorbitol as supplementary carbon source in the induction phase, it effectively improve the AOX enzyme activity and the cell survival rate, HLZ activity enhanced7.6%in the shake flasks culture and12.3%in the5L fermentation tank culture compared to the sole methanol feeding. Examines the effect of the initial cell concentration of induction phase and dissolved oxygen control strategy to the expression of HLZ, it found that keep the initial cell concentration at100g/L and maintain the dissolved oxygen between5-15%in the induction phase is good for the expression.The use of Fourier transform mid-infrared spectroscopy (FTIR) combined with PLS to predict the concentrations of glycerol and methanol in fed-batch cultivations of Pichia pastoris in a chemical medium was investigated, by using the synthetic solutions as calibration set. Generally, all models were robust and performed well, the calibration model allowed standard errors of calibration (SEC) and standard errors of validation (SEV) for glycerol and methanol concentrations of0.171,0.532,0.129and0.248g/L, respectively.5L Fed-batch experiments show that FTIR data has a good agreement with control date, with standard error of prediction (SEP) of1.09and0.86g/L, respectively. Specific growth rate and specific production rate was compared between FTIR and DO-stat to control methanol concentration. It was showed that FTIR can limit the methanol concentration under20g/L which can maintain a higher specific growth rate and specific production rate that was effective to enhance the production.
Keywords/Search Tags:HLZ, response surface methodology, sorbitol, osmotic stress, FTIR
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