Functional Enzyme Expression in E.coli
We then evaluated functional enzyme expression in E. coli (enzyme provided by Evocatal). Expression in rich TB media and synthetic WR media containing 20 g/L glucose revealed that cultivation in Duran baffled flasks was better than that in classical Duran Erlenmeyer flasks. In the latter ones the lower oxygen-transfer capacity led to a higher formation of acidic byproducts.
In synthetic WR media, the pH value decreased very fast below a pH of 5, as a result cell growth was inhibited. With Duran baffled flasks, higher cell densities were obtained because of slower and reduced acidification in the culture broth. The metabolization of complex compounds in rich TB media results in rising pH value during cultivation subsequent to consumption of glycerol as the main carbon source. In classical Duran Erlenmeyer flasks a higher rate of acidic byproduct formation can be observed by a slower rate of pH increase.
The better oxygen-transfer conditions exhibited with Duran baffled flasks can also be observed in higher expression of the target enzyme. Activities in WR medium differ by one order of magnitude because cells cultivated in Erlenmeyer flasks are faced with an acidic surrounding, which prevents growth and significant product formation.
Further study was conducted by using FIT fed-batch media (synthetic and complex formulation) both in Duran baffled flasks and Duran Erlenmeyer flasks.
The maximum applicable glucose feeding rate is dependent on the oxygen-transfer capacity of the bioreactor vessel. With this in mind, two concentrations of glucose-releasing enzyme were investigated. As shown in Figure 2, both glucose feeding rates could be realized in Duran baffled flasks. For the higher glucose release rate (1.5% enzyme) in the classical Erlenmeyer flasks, however, the culture was not able to consume the total fed glucose due to limited oxygen in these flasks.
As a result, glucose accumulated in the broth, and cells switched to an overflow metabolism and started to produce acidic byproducts. As a consequence the pH value quickly dropped to a growth-inhibiting value of 5. In addition, productivity was dramatically decreased under these conditions.
In Duran baffled flasks the cells experienced a higher oxygen supply and, therefore, they were able to consume the fed glucose at the same rate as it was released for the higher release rate with 1.5% enzyme. Consequently, this led to faster generation of biomass (=50 OD600 after 24 hours) and higher target enzyme expression.
When comparing enzyme expression in batch and fed-batch mode, the target enzyme exhibited greater activity in fed-batch fermentation, and the proportion of active product to inactive product was improved. This means that the growth-limiting glucose feeding in FIT fed-batch medium improves the overall product formation and also produces more active protein in the soluble fraction.