Integrated flow-injection processing for on-line quantification of plasmid DNA during cultivation of E. coli.

An integrated flow-injection processing (FIP) system for the quantification of plasmids during cultivation is described. The system performs on-line sampling, cell lysis, and quantification of plasmids in an integrated manner during cultivation of E. coli. The system was operated by using a miniaturized expanded-bed column which can be used for handling samples containing cells and cell debris without interfering with the binding analysis. Two types of detectors (one measuring UV absorbance at 254 nm and a fluorometer) are used for on-line plasmid detection. The system was developed using standard solutions and it was successfully applied in monitoring plasmid contents during a cultivation of E. coli.

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.

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.

Evidence for substrate binding of a recombinant thermostable xylanase originating from Rhodothermus marinus.

The xynl encoded 5 domain xylanase from the thermophilic bacterium Rhodothermus marinus binds specifically to xylan, beta-glucan and amorphous but not crystalline cellulose. Our results show that the binding is mediated by the full length xylanase, but not by the catalytic domain only. Based on similarities concerning both predicted secondary structure and binding specificity found with one cellulose binding domain of CenC from Cellulomonas fimi, we suggest that the binding is mediated by the two N-terminally repeated domains.

Enzymatic specificity and hydrolysis pattern of the catalytic domain of the xylanase Xynl from Rhodothermus marinus.

The catalytic domain of a xylanase from Rhodothermus marinus was produced in Escherichia coli. The catalytic domain belongs to glycosyl hydrolase family 10. The produced protein has a 22-amino acid leader peptide followed by a 411-amino acid truncated xylanase. The molecular mass was 48 kDa and the recombinant xylanase had a pI of 4.9. The pH and temperature optima for activity were determined to be 7.5 and 80 degrees C, respectively. At that temperature the enzyme had a half-life of 1 h 40 min. An addition of 1 mM calcium stabilized the activity of the enzyme at 80 degrees C. The xylanase had its highest specific activity on oat spelt xylan but was active also on other xylans and to a limited extent on some other polysaccharides (soluble glucans). No exo- or endo-cellulase activity was observed. Hydrolysis of xylo-oligomers and oat spelt xylan was studied and the predominant products of hydrolysis were xylobiose and xylotriose. The enzyme was inactive on xylobiose, xylotriose and on the soluble fraction from oat spelt xylan. The R. marinus xylanase is shown to have a strong preference for internal linkages and is therefore classified as an endo-xylanase.