Secretory production of Arthrobacter levan fructotransferase from recombinant Escherichia coli

Levan fructotransferase (LFTase) from Arthrobacter ureafaciens K2032 was ex pressed with N-terminal fusion of a LacZ-derived secretion motif (TMITNSSSV P) using the lac promoter system in recombinant Escherichia coli JM109 [pUD F-A81]. In flask cultures, recombinant enzyme activity was detected in cult ure media, and sequence analysis of N-terminal residues showed that about 4 0% of the extracellular recombinant LFTase had an authentic N-terminus. In a fed-batch bioreactor containing recombinant E. coli at high cell concentr ations (OD600 > 200), the extracellular LFTase accumulated to 46 000 U ml(- 1) (similar to 2.0 g l(-1)) which was almost 40% of total (intra- and extra cellular) recombinant LFTase. The synthesized recombinant enzyme was secret ed soon after gene expression was induced by IPTG. Prolonged high secretion caused cell lysis and growth inhibition during the production phase in fed -batch cultures. When lactose was added by continuous feed mode, the secret ion of recombinant LFTase and hence the cell lysis were significantly delay ed in spite of the increased synthesis level. Therefore the induced cell cu lture of recombinant E. coli could grow up to a much higher cell concentrat ion with continuing recombinant enzyme synthesis. In the case of the contro lled feed of lactose, the maximum activities (U ml(-1)) of total and extrac ellular LFTase were nearly 100% and 70% higher, respectively. (C) 2001 Fede ration of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.