On-line detection of acetate formation in Escherichia coli cultures using dissolved oxygen responses to feed transients.

Recombinant protein production in Escherichia coli can be significantly reduced by acetate accumulation. It is demonstrated that acetate production can be detected on-line with a standard dissolved oxygen sensor by superimposing short pulses to the substrate feed rate. Assuming that acetate formation is linked to a respiratory limitation, a model for dissolved oxygen responses to transients in substrate feed rate is derived. The model predicts a clear change in the character of the transient response when acetate formation starts. The predicted effect was verified in fed-batch cultivations of E. coli TOPP1 and E. coli BL21(DE3), both before and after induction of recombinant protein production. It was also observed that the critical specific glucose uptake rate, at which acetate formation starts, was significantly decreased after induction. On-line detection of acetate formation with a standard sensor opens up new possibilities for feedback control of substrate feeding.

Use of a micro-expanded bed containing immobilised lysozyme for cell disruption in flow injection analysis.

A method for cell disruption in Flow Injection Analysis (FIA) systems has been developed. The principle involves on-line cell disruption by means of immobilised lysozyme followed by an ultrasonic treatment. In order to avoid flow problems in the analytical system, the lysozyme was immobilised to Streamline that was used in an expanded bed in the flow system. Samples of suspensions of Micrococcus lysodeikticus were treated and the success of the treatment was evaluated in terms of released protein and as a decrease in the optical density at 450 nm. The new technology offers a powerful tool in flow injection analyses for quantification of intracellular compounds. The concept of integration, i.e. combining cell disruption with handling of cell debris and assay procedure in one continuous flow process facilitates its use and increases the probability of reaching reproducible and reliable results.

Flow injection analysis of intracellular beta-galactosidase in Escherichia coli cultivations, using an on-line system including cell disruption, debris separation and immunochemical quantification.

A continuous integrated process for on-line quantification of intracellular components has been developed. By applying the concept of expanded micro-beds in a flow injection system it was possible to first perform on-line cell disintegration followed by an on-line binding assay for quantification of a reporter protein (beta-galactosidase) from the cell interior. The disintegration process involved the use of an expanded bed with immobilised lysozyme followed by ultrasonic treatment in a flow-through cell. The cell debris does not interfere in the binding assay as it is carried out in an expanded bed. The time for an assay cycle is at present approx. 35 min. This integrated system can be used for quantification of proteins down to at least 10(-7) mol/L.

Efficient production of truncated thermostable xylanases from Rhodothermus marinus in Escherichia coli fed-batch cultures.

A cultivation strategy for the production of two truncated thermostable recombinant xylanases (Xyn1deltaN and Xyn1deltaNC) was developed. Fed-batch cultivations of Escherichia coli strain BL21(DE3) with a controlled exponential glucose feed led to high specific production of the recombinant proteins. Addition of complex nutrients (e.g. Tryptone Soya Broth (TSB)) to the media were shown to increase both the specific growth rate during the production phase and the production per cell. The final cell-mass concentration depended on the time of induction in relation to both the feed-start and the expected time at which the cultivation had to be terminated due to oxygen transfer limitations or cell lysis. The gene used for the genetic constructions (encoding Xyn1deltaN and Xyn1deltaNC) was originally isolated from Rhodothermus marinus. Recombinant protein expression was controlled by the T7 lac-promoter and induced in the fed-batch phase at low glucose concentrations by the single addition of either lactose or isopropyl-thio-beta-d-galactoside (IPTG). In lactose-induced cells, the production of recombinant xylanase was delayed for approximately 30 min in comparison with those induced with IPTG, but the specific product levels were comparable at 3 h after induction. At this time, approximately 35% of the intracellular protein content was constituted by recombinant xylanase. Under the cultivation conditions used, production of the shorter deletion derivative (Xyn1deltaNC) led to nonspecific leakage and cell lysis, starting 1.5 or 2 h after induction with IPTG or lactose, respectively. At 3 h after induction, 50% of the produced protein (Xyn1deltaNC) was found in the culture medium. This was not the case for the longer protein (Xyn1deltaN), where only 10% of the xylanase leaked into the medium.