Display of active enzymes on the cell surface of Escherichia coli using PgsA anchor protein and their application to bioconversion.
We have developed a novel Escherichia coli cell surface display system by employing PgsA as an anchoring motif. In our display system, C-terminal fusion to PgsA anchor protein from Bacillus subtilis was used. The enzymes selected for display were alpha-amylase (AmyA) from Streptococcus bovis 148 and lipase B (CALB) from Candida antarctica. The molecular mass values of AmyA and CALB are approximately 77 and 34 kDa, respectively. The enzymes were displayed on the surface as a fusion protein with a FLAG peptide tag at the C terminus. Both the PgsA-AmyA-FLAG and PgsA-CALB-FLAG fusion proteins were shown to be displayed by immunofluorescence labeling using anti-FLAG antibody. The displayed enzymes were active forms, and AmyA and CALB activities reached 990 U/g (dry cell weight) and 4.6 U/g (dry cell weight), respectively. AmyA-displaying E. coli cells grew utilizing cornstarch as the sole carbon source, while CALB-displaying E. coli cells catalyzed enantioselective transesterification, indicating that they are effective whole-cell biocatalysts. Since a target enzyme with a size of 77 kDa and an industrially useful lipase have been successfully displayed on the cell surface of E. coli for the first time, PgsA protein is probably a useful anchoring motif to display various enzymes.
Narita
Junya
J
Division of Molecular Science, Graduate School of Science and Technology, Kobe University, Nada-ku, Kobe, 657-8501, Japan.
Okano
Kenji
K
Tateno
Toshihiro
T
Tanino
Takanori
T
Sewaki
Tomomitsu
T
Sung
Moon-Hee
MH
Fukuda
Hideki
H
Kondo
Akihiko
A
D002175:Candida / Q000201:enzymology; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000166:cytology* / Q000378:metabolism*; D005656:Fungal Proteins; D015870:Gene Expression; D008049:Lipase / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics / Q000378:metabolism*; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism
D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D017855:Transferases (Other Substituted Phosphate Groups); C020676:CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase; D008049:Lipase; C431349:lipase B, Candida antarctica; D000516:alpha-Amylases