Fermentation Kinetics Of Producting Recombination Human-like Collagen In Pichia Pastoris

Collagen is widely used in various fields, such as food, hairdressing, medical and health, etc., because of its good biological compatibility, decomposability and low immunogenic idiosyncrasy. Response Surface Methodology (RSM) was used to optimize the fermentation process of recombinant Pichia pastoris (GS115/pPIC9KG6) expressing human-like collagen which was constructed in our laboratory.Box-Behnken Design was introduced to estimate the impact of initial inducement pH, temperature and the accession quantity of methanol on expressing human-like collagen in recombinant Pichia pastoris. The result of RSM analysis showed that the optimal scale of factors is: initial inducement pH 4.7, temperature 28℃and the accession quantity of methanol 2.1%/24h. The kinetics of fed-batch fermentation in 3.7L fermenter (Bioengineering KLF2000) of recombinant Pichia pastoris was investigated. And a non-structure model of microorganism growth, a production formation model and a limited substrate (glycerol and methanol) consumption model were obtained to optimize the fermentation process.SDS-PAGE analysis of the intracellular and extracellular production showed that human-like collagen could be expressed both in and out of the cell. Furthermore, electrophoresis degree of human-like collagen was obtained through purifying the fermentation supernatant by anion exchange chromatography and gel filtration chromatography. Through Analyzing the ultraviolet-visible spectroscopy and FTIR of recombinant human-like collagen, we presume that purified product is similar to native collagen in protein structure.