Secretory production of Arthrobacter levan fructotransferase from recombinant Escherichia coli.
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 (OD(600)>200), the extracellular LFTase accumulated to 46000 U ml(-1) (approximately 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.
Lee
J
J
Microbial and BioProcess Engineering Laboratory, Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yusong, Taejon 305-600, South Korea. jwlee@mail.kribb.re.kr
Saraswat
V
V
Koh
I
I
Song
K B
KB
Park
Y H
YH
Rhee
S K
SK
D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000201:enzymology* / Q000235:genetics; D019149:Bioreactors; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D006602:Hexosyltransferases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D007785:Lactose / Q000378:metabolism; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry
D011993:Recombinant Fusion Proteins; D007544:Isopropyl Thiogalactoside; D006602:Hexosyltransferases; C040074:levan fructotransferase; D007785:Lactose