Recombinant Protein Production by Escherichia coli BL21 (DE3) [pET-12-STA1] Using a Bioreactor with Cross-Flow Filtration.

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  • Kobayashi Fumihisa
    Department of Chemistry and Chemical Engineering, Faculty of Engineering, Kanazawa University
  • Nakamura Yoshitoshi
    Department of Chemistry and Chemical Engineering, Faculty of Engineering, Kanazawa University

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In order to produce a recombinant protein rapidly and efficiently, glucoamylase, as a recombinant protein, was produced by Escherichia coli BL21 (DE3) [pET-12-STA1] using a bioreactor with cross-flow filtration. The recombinant E. coli BL21 (DE3) [pET-12-STA1] having an overexpression system with a recombinant plasmid (pET-12-STA1) was constructed by inserting a STA1 gene (a glucoamylase gene) into an overexpression vector (pET-12). Glucoamylase activity decreased rapidly after an incubation time of 2 h due to the degradation of glucoamylase by a protease synthesized with cell growth. The molecular weights of glucoamylase and protease were about 80 kDa and 10 kDa by gel filtration chromatography. Therefore, the culture using a bioreactor with cross-flow filtration was attempted for the separation of glucoamylase from the medium containing a protease. The filtration for enzyme separation was used by ultrafiltration membrane having a nominal molecular weight cutoff of 50 kDa. The glucoamylase activity obtained using the bioreactor with cross-flow filtration reached about 20 U cm–3 and this value was much higher than that without cross-flow filtration, i.e. 6.6 U cm–3. Since the separated liquid included both glucoamylase and protease, a recycle-type bioreactor with a cross-flow filtration system was also devised for the separation of glucoamylase from the separated liquid. The glucoamylase activity obtained using a recycle-type bioreactor system was maintained at about 40 U cm–3 and this value was about 2-fold higher than that without recycling. The recycle-type bioreactor system could produce 17220 U glucoamylase in about 1 dm3 culture medium; it was about 2 times higher than that without recycling (8700 U).

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