Secretory production of Arthrobacter levan fructotransferase from recombinant Escherichia coli

Jeewon Lee, Vibhor Saraswat, Isaac Koh, Ki Bang Song, Young Hoon Park, Sang Ki Rhee

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Levan fructotransferase (LFTase) from Arthrobacter ureafaciens K2032 was expressed with N-terminal fusion of a LacZ-derived secretion motif (TMITNSSSVP) using the lac promoter system in recombinant Escherichia coli JM109 [pUDF-A81]. In flask cultures, recombinant enzyme activity was detected in culture media, and sequence analysis of N-terminal residues showed that about 40% of the extracellular recombinant LFTase had an authentic N-terminus. In a fed-batch bioreactor containing recombinant E. coli at high cell concentrations (OD600>200), the extracellular LFTase accumulated to 46 000 U ml-1 (∼2.0 g l-1) which was almost 40% of total (intra- and extracellular) recombinant LFTase. The synthesized recombinant enzyme was secreted 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 secretion of recombinant LFTase and hence the cell lysis were significantly delayed in spite of the increased synthesis level. Therefore the induced cell culture of recombinant E. coli could grow up to a much higher cell concentration with continuing recombinant enzyme synthesis. In the case of the controlled feed of lactose, the maximum activities (U ml-1) of total and extracellular LFTase were nearly 100% and 70% higher, respectively.

Original languageEnglish
Pages (from-to)127-132
Number of pages6
JournalFEMS microbiology letters
Volume195
Issue number2
DOIs
Publication statusPublished - 2001 Feb 20
Externally publishedYes

Keywords

  • Escherichia coli
  • High-cell-density culture
  • Levan fructotransferase
  • Secretion

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology
  • Genetics

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