Efficient production of truncated thermostable xylanases from Rhodothermusmarinus in Escherichia coli fed-batch cultures

A cultivation strategy for the production of two truncated thermostable rec ombinant xylanases (Xyn1 Delta N and Xyn1 Delta NC) was developed. Fed-batc h cultivations of Escherichia coil strain BL21(DE3) with a controlled expon ential glucose feed led to high specific production of the recombinant prot eins. Addition of complex nutrients (e.g. Tryptone Soya Broth (TSB)) to the media were shown to increase both the specific growth rate during the prod uction phase and the production per cell. The final cellmass concentration depended on the time of induction in relation to both the feed-start and th e expected time at which the cultivation had to be terminated due to oxygen transfer limitations or cell lysis. The gene used for the genetic construc tions (encoding Xyn1 Delta N and Xyn1 Delta NC) was originally isolated fro m Rhodothermus marinus. Recombinant protein expression was controlled by th e T7 lac-promoter and induced in the fed-batch phase at low glucose concent rations by the single addition of either lactose or isopropyl-thio-beta-D-g alactoside (IPTG). In lactose-induced cells, the production of recombinant xylanase was delayed for approximately 30 min in comparison with those indu ced with IPTG, but the specific product levels were comparable at 3 h after induction. At this time, approximately 35% of the intracellular protein co ntent was constituted by recombinant xylanase. Under the cultivation condit ions used, production of the shorter deletion derivative (Xyn1 Delta NC) le d to nonspecific leakage and cell lysis, starting 1.5 or 2 h after inductio n with IPTG or lactose, respectively. At 3 h after induction, 50% of the pr oduced protein (Xyn1 Delta NC) was found in the culture medium. This was no t the case for the longer protein (Xyn1 Delta N), where only 10% of the xyl anase leaked into the medium.