Fermentation Optimization And S-adenosyl-L-methionine Separation From Recombinant Pichia Pastoris

S-adenosyl-L-methionine (SAM) is an important metabolic intermediate in all livingorganisms, which is utilized by transmethylation, transsulfuration, and polyamime sythesis.An HPLC method for determination of SAM was established. The analysis method wasperformed witha C18column (150mm×4.6mm,5μm) at30°C, the mobile phase consisted of97%0.01mol/L ammonium formate (pH3.5) and3%methanol, flow rate0.6mL/min, anddetection wavelength260nm. At the same time, two methods for determination of methanolconcentration by methanol sensor were estabished: the silicone tubing sensor and sensorprototypes measuring methanol concentrations in the exhaust gas.The effects of L-methionine and methanol concentration on cell growth and SAMproduction were studied in shake-flask culture by Pichia pastoris. After optimization, SAMproduction reached0.81g/L when initial L-methionine concentration was7.5g/L at shake flask.Moreover, SAM production could reach1.41g/L when methanol concentration was controlled at15g/L during the fermentation, increased by21%more than that of the control condition.Although glycerol is a repressor to the AOX1promoter, it is a most popular substrate usedin the Pichia pastoris fermentation process.With a feeding strategy using10%glycerol-methanolas a carbon source,6.09g/L of SAM could be produced. Two sets of glycerol-methanolmixed-feeding strategy were performed at the7L fermentor, the maxium SAM reached9.79g/L.At last, yeast cells were disrupted and intracellular SAM was extracted by repeated frozenmethod at pH3.0, and then SAM was separated from the crude extract by weak-acid caiomexchange column chromatography. The recovery rate of SAM reached74.7%during the wholeseparation process.