Effects of fermentation conditions on the production of 4-α-glucanotransferase from recombinant Escherichia coli

Abstract

4-α-Glucanotransferase (4-α-GTase, EC 2.4.1.25) simultaneously catalyzes amylose molecular intra- and inter- transglycosylation to produce cyclodextrins (CDs) with DP of 9 to several hundreds. In this study, the 4-α-glucanotransferase (4-α-GTase) from recombinant Escherichia coli DH-5α was proved to produce large ring cyclodextrins (LR-CDs) with polymer degree of 20 to 47. To maximize the 4-α-GTase productivity effect, conditions for the production of 4-α-GTase were initially carried out in shake flasks to investigate the effects of carbon sources, nitrogen sources, trace elements, inoculum size, liquid volume, pH and temperature. The results showed that the production of 4-α-GTase and increase of biomass (E. coli growth) was out of sync. Glycerol of 20 g/l in medium, as the best carbon source, provided as high as 102.55 U/ml of 4-α-GTase, which was 0.59-fold compared with the minimal medium. Organic nitrogen source enhanced more enzyme activity than the inorganic source; beef extract was identified as the most effective source for enzyme activity enhancement. In order to get the highest enzyme activity, the optimum incubation conditions were initial pH of fermentation broth at 7.0, culture temperature of 37°C, inoculum size of 2.0% and liquid volume of 50 ml in 250 ml Erlenmeyer. Under these conditions, 4-α-Gtase activity in 5 L fermenter reached as high as 198.7 U/ml.

Key words: High cell density, 4-α-glucanotransferase, recombinant Escherichia coli, large-ring cyclodextrin, optimum incubation conditions