Production And Purification Of Capsular Polysaccharide By S.Aureus With Liquid Fermentation

Staphylococcus aureus (S. aureus) is an opportunistic bacterial pathogen responsible for a diverse spectrum of human and animal diseases. One of its major infectious agents is capsular polysaccharides (CPs), which envelope the surface of S. aureus. In this research, S. aureus which synthesize type5capsular polysaccharides was chosen as parent strain. In order to acquire a great quantity of bacteria and CP by means of high density fermentation, we optimized the growth conditions of S. aureus in shake flask and in5liter bioreactor. We also tried to extract and purify CPs from the bacteria.In this research, concentration of lactose and yeast extract, initial pH, volume of medium on bacteria quantities and CP production were studied. Optimal fermentation conditions of S. aureus in shake flask were as follows:tryptone1.0%(w/v), NaCl0.5%(w/v), lactose0.2%(w/v), yeast extract0.4%(w/v), initial pH7.5, temperature37℃,180rpm, working volume100ml/500ml, cultivation time10h. Under these conditions, OD600could reach as much as2.52, the production of polysaccharide could reach32.77mg/L, which were1.46times and3.29times than that of initial conditions.Optimal fermentation conditions of S. aureus in5liter bioreactor were as follows: glutamic acid1.3g/L, cystine12mg/L, MgCl26H2O0.4g/L,Na2HPO4·12H2O5.7g/L, NH4C16g/L, KC190mg/L, NaCl6g/L, lactose2g/L, yeast extract4g/L, nicotinamide lmg/L, pH7.5, ventilator capacity3L/min, rotate speed100rpm, cultivation time36h. Under these conditions, OD600could reach as much as5.05, the production of polysaccharide could reach56.67mg/L, which were2times and1.73times than that in shaking-flask fermentation respectively.By the analysis of fermentation in5liter bioreactor with S. aureus, fed batch culture was used for further increasing CP production. Fed batch culture conditions were as follows: fermentation medium containing20%lactose was added to the bioreactor at a speed of15ml/h (3g lactose/h). Total feeding time was66h, and198g lactose was used. OD600could reach as much as9.43, and the production of polysaccharide could reach101.49mg/L, which were3.74times and3.1times than that in shaking-flask fermentation respectively.S. aureus cultivated in5L bioreactor are treated as follows:the strains were autoclaved, the supernatants were precipitated by CTAB, dissociated by CaCl2, precipitated by30%(v/v) ethanol and then80%(v/v) ethanol, nucleic acid was hydrolysed by DNase and RNase, protein was hydrolysed by pronase, teichoic acid was oxidized by NaIO4, dialyzed and lyophilized. Lyophilization was applied to DEAE-FF in order to acquire purified CP.Liquid fermentation of S. aureus which can acquire more CP than solid fermentation was conducive to realize commercial production of CP conjugate vaccine. Some cases were very important for separation and purification CP, such as reducing CP lose, wiping off as much impurities as possible, using special method of detecting CP.