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

• Published in: Journal of bioscience and bioengineering Vol. 87; no. 5; pp. 598 - 606
• Main Authors: Karlsson, Eva Nordberg, Holst, Olle, Tocaj, Anita
• Format: Journal Article
• Language: English
• Published: Amsterdarm Elsevier B.V 01.01.1999; Elsevier Science
• Subjects: BACTERIA; Biological and medical sciences; Bioprocess Technology; Bioprocessteknik; Biotechnology; Complex nutrients; Engineering and Technology; Enzyme engineering; Fermentation; Fundamental and applied biological sciences. Psychology; HIDROLASAS; HYDROLASE; HYDROLASES; Industrial Biotechnology; Industriell bioteknik; isopropyl-thio-β- d-galactoside (IPTG); Lactose; Methods. Procedures. Technologies; MICROORGANISME THERMOPHILE; MICROORGANISMOS TERMOFILOS; Production of selected enzymes; PROTEINAS RECOMBINANTES; PROTEINE RECOMBINANTE; RECOMBINANT PROTEINS; Recombinant xylanase; Teknik; THERMOPHILIC MICROORGANISMS; isopropyl-thio-β- d-galactoside (IPTG); lactose; complex nutrients; recombinant xylanase; Culture medium; Enzyme; Induction; Escherichia coli; Glucose; Gene expression; Thermal stability; Lactose; Regulation(control); Glycosidases; Production; Microorganism culture; Deletion; Bacteria; Hydrolases; Xylan endo-1,3-β-xylosidase; Mutation; Recombinant protein; O-Glycosidases; Semicontinuous; Enterobacteriaceae
• ISSN: 1389-1723; 1347-4421
• DOI: 10.1016/S1389-1723(99)80121-2

A cultivation strategy for the production of two truncated thermostable recombinant xylanases (Xyn1ΔN and Xyn1ΔNC) 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 cellmass 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 Xyn1ΔN and Xyn1ΔNC) 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-β- 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 (Xyn1ΔNC) 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 (Xyn1ΔNC) was found in the culture medium. This was not the case for the longer protein (Xyn1ΔN), where only 10% of the xylanase leaked into the medium.