Fermentation Optimization Of α-cyclodextrin Glycosyltransferase Production Mediated By Ompa Singal Peptide In Recombinant E.coli

Cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) is capable of converting starch or starch derivates into cyclodextrins (CDs) through an intramolecular transglycosylation reaction. The most common forms of CDs areα-,β- andγ-CDs, among whichα-CD has wider applications in molecular recognition, nano materials, and food industries for its better solubility and smaller internal cavity. Thus,α-CGTase has become the focus of scientific research nowadays.Previously, our laboratory achieved the extracellular production ofα-CGTase from Paenibacillus macerans JFB05-01 in recombinant Escherichia coli BL21 (DE3) with OmpA as signal peptide. With the aim to achieve the high yield and productivity ofα-CGTase in the medium by scale-up fermentation, different media and culture conditions on the extracellular production ofα-CGTase were investigated in shaking flasks and 3-L fermentators. The main results are listed as follows:1,The optimal seed age of recombinant E. coli was 6 to 8h. The addition of 0.75%(w/v) glycine during the pre-logarithmic phase in shaking flasks could effectively improve the extracellularα-CGTase production and the enzyme activity reached 18.9 U/mL at 36h of the culture, which was 2.2-fold as compared with that of control. The required nutrients and conditions forα-CGTase production by E. coli in shaking flasks were investigated with a series of single-factor experiments. The optimal fermentation medium and culture condition were as follows:10 g/L glycerol,18 g/L peptone,20 g/L yeast extract,16.43 g/L, K2HPO4·3H2O,2.31 g/L KH2PO4,5mM MgCl2, an initial pH of 7.0, a temperature of 30℃, an inoculum size of 7% and a medium content of 20 mL medium in 250 mL flask. After optimization, the extracellular production ofα-CGTase was enhanced from 20.5 U/mL in 60h to 56.0 U/mL in 48h, and the productivity from 341.7 U/L/h to 1166.7 U/L/h.2,The effects of dissolved oxygen (DO) concentrations on the batch fermentation ofα-CGTase by E. coli in 3-L fermentor were investigated. It was found when cultured under 30% DO concentration, the extracellular activity reached 17.76 U/mL, and it was 1.7 and 1.2-fold of that under 20% DO and 40% DO concentration, respectively. The effects of different inducers onα-CGTase production by fed-batch cultivation were studied. The results showed thatα-CGTase production under induced conditions were higher than that of non-induced conditions. When induced by IPTG, the extracellular activity reached 49.74 U/mL, which was 1.6-fold as compared with that of non-induced conditions. When induced by lactose, the extracellular activity reached 44.0 U/mL, which was 1.4-fold as compared with that of non-induced conditions.3,The effect of synthetic medium on high cell density culture ofα-CGTase production by E. coli in 3-L fermentor were investigated under non-induced conditions. The results showed that the synthetic medium was suitable for high cell density culture, in which the cell reached the highest OD600 of 150.0, but none activity were produced under non-induced conditions. In order to induce the recombinant protein production by E.coli, different inducers, concentration of lactose and induction points were investigated on the high cell density culture of E.coli in 3-L fermentor. The results showed that 1) none activity could be detected in the medium for its autolysis after 1h of 0.5 mM IPTG induction. However, the activity induced by 8.0 g/L/h lactose was 10.0 U/mL in the medium.2) The activity induced by 0.8 g/L/h lactose gained 46.44 U/mL in the medium at 27h, which was 1.8 and 4.6-fold as compared with that induced by 0.4 and 8.0 g/L/h lactose.3) When induced at OD600 of 50, the extracellular activity was 5.5 and 66.3-fold of that under induction at OD600 of 25 and 100.5,Different nitrogen in the feeding solution and induction temperature were investigated on the high cell density culture of E.coli in 3-L fermentor. It was observed that 1) the extracellular activity under organic nitrogen addition in the feeding solution was 105.1 U/mL, which was 2.3-fold of that of control. The extracellular activity under inorganic nitrogen addition in the feeding solution was 8.8 U/mL, accounting 19.0% of the control. The extracellular activity under induction temperature of 25℃gained 275.3 U/mL in the medium at 35h, which was 5.1 and 3.6-fold of that under 20℃and 30℃.