| Rhizomucor miehei lipase (RML) is one of the most widely used lipases produced by microorganism. RML can catalyze greases hydrolyze, the reaction of ester synthesis, transesterification and acidolysis in the non-aqueous phase. The cost in the bioprocess of this enzyme production can be efficiently reduced by displaying RML on Pichia pastoris cell surface, which shows the characteristics of the immobilized enzyme. Complicated purification process unnecessary, the cells can be easily recycled during the catalytic reaction. Although the production level of a heterologous protein is mainly depended on the inherent properties of strains, the gene doesage and stability of the heterologous protein. Enhancement of productivity can also be made by optimizing fermentation methodology.In this study, fermentation batches were carried out in 50L scale fermentor, to investigate the factors that influence the production of RML displayed on Pichia pastoris cell surface. Different culture conditions were studied. Flow cytometry technology was implemented to detect and analyze the cell viability and RML production displayed on cell surface. Various environmental conditions that affect the production of RML were analyse as well, by studying the change trend of the online metabolic parameters, such as OUR,CER and RQ。The study indicated that the optimal percentage of liquid volume in shake flask is 10%, while too much volume can resulted in limited oxygen, which actually influences the production of RML in bioreactor culture.When cell density (OD600) before induction is 268, enzyme activity of RML displayed on cell surface and productivity achieved maximum, that is 255.6 U/g and 456.3 U/(L·h), respectively. This illustrates that relative lower initial induction cell density contributes to the production of RML in Pichia pastoris compared to that of higher cell density.Pichia pastoris can grow under the condition of pH ranging from 3.0 to 7.0. However, the optimal pH is 6.0, under which RML activity peaked at 275.0U/g, 54.5% higher than that under initial conditions.When the induction temperature was set at 20℃and 25℃, methanol transformation efficiency are 1.56 and 1.32 folds as high as that at 30℃. This result shows that reducing induction temperature helps to increase methanol transformation efficiency and increase productivity efficiently.Methanol/Sorbitol co-feeding strategy was also studied. By implementing this strategy, the RML productivity at t=12h was 1.7-fold as high as that by sole carbon resource, when the sorbitol and methanol mix rate was set at 1:4 and 1:5.Methods were identified to detect Pichia pastoris cell viability and the amount of RML displayed on cell surface during fermentation process. Data shows that unlike other conditions, when induction pH was set at 7.0, decrease of enzyme activity of RML was observed after t=62 h. In this case, cell viability was detected by flow cytometry technology, from which saw a continual decrease of cell viability after induction, while under other conditions cell viability maintained above 99%. This maybe resulted from cell lysis, and protease was released, which cause protein proteolysis. Flow cytometry technology was also implemented to analyse the production of RML displayed on cell surface. The study showed that fluorescence intensity is relative to RML enzyme activity, and the amount of RML on the surface is relative to fluorescence intensity.OUR,CER and RQ are very important parameters correlated to the metabolic activity in the organism. In this study, the same change trend can be observed in OUR and CER under different conditions, except under the condition of low temperature during induction phase. Under 20℃, the OUR and CER only maintained at around 110 mmol l-1h-1 and 60 mmol l-1h-1,only 44% compared to that under 30℃, while under 25℃, OUR and CER were 130 mmoll-1h-1and70 mmoll-1h-1, 52% to that under 30℃. In addition, study indicates that during batches with higher RML enzyme activity, RQ value maintained between 0.50-0.55, while it maintained between 0.55-0.65 during batches with lower RML activity.
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