title: A sensitive single-enzyme assay system using the non-ribosomal peptide synthetase BpsA for measurement of L-glutamine in biological samples.
abstract: The ability to rapidly, economically and accurately measure L-glutamine concentrations in biological samples is important for many areas of research, medicine or industry, however there is room for improvement on existing methods. We describe here how the enzyme BpsA, a single-module non-ribosomal peptide synthetase able to convert L-glutamine into the blue pigment indigoidine, can be used to accurately measure L-glutamine in biological samples. Although indigoidine has low solubility in aqueous solutions, meaning direct measurements of indigoidine synthesis do not reliably yield linear standard curves, we demonstrate that resolubilisation of the reaction end-products in DMSO overcomes this issue and that spontaneous reduction to colourless leuco-indigoidine occurs too slowly to interfere with assay accuracy. Our protocol is amenable to a 96-well microtitre format and can be used to measure L-glutamine in common bacterial and mammalian culture media, urine, and deproteinated plasma. We show that active BpsA can be prepared in high yield by expressing it in the apo-form to avoid the toxicity of indigoidine to Escherichia coli host cells, then activating it to the holo-form in cell lysates prior to purification; and that BpsA has a lengthy shelf-life, retaining >95% activity when stored at either -20 °C or 4 °C for 24 weeks.
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand.
authors.forename: Alistair S
authors.initials: AS
authors.lastname: Brown
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand.
authors.forename: Katherine J
authors.initials: KJ
authors.lastname: Robins
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand.|Centre for Biodiscovery, Victoria University of Wellington, Wellington, New Zealand.
authors.forename: David F
authors.initials: DF
authors.lastname: Ackerley
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015415:Biomarkers; D057075:Enzyme Assays / Q000379:methods; D015870:Gene Expression; D005973:Glutamine / Q000378:metabolism*; D010453:Peptide Synthases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010881:Piperidones / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D001426:Bacterial Proteins; D015415:Biomarkers; D010881:Piperidones; D005973:Glutamine; C446045:indigoidine; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase