A bioluminescent Escherichia coli auxotroph for use in an in vitro lysine availability assay

Microbiological methods have been used to determine the amino acid availabi lity of a variety of animal feed and human food protein sources. Growth of Escherichia coli auxotrophs have been shown to yield a consistent linear re sponse to lysine concentration when compared to chemical measures. Extent o f total growth of E. coli lysine mutant (American Type Culture Collection # 23812) when measured as optical density (OD) displays a lysine-dependent gr owth response that can be used to estimate lysine in feed proteins. However , typical OD-based growth studies for amino acid quantitation using the mut ant may require anywhere from 12 to over 40 h. To develop an improved rapid method for lysine quantitation in protein sources, the plasmid pJHD500 car rying genes that encode for expression of bioluminescence and ampicillin re sistance was transformed into the E. coli mutant by electroporation (set at 1.80 kV). The luminescence measured during early exponential growth allowe d detectable differentiation of lysine concentration in the media in 4 h. W hen the luminescence method was compared with the conventional optical dens ity lysine growth assay, the correlation coefficient was 0.989. Lysine avai lability valued for enzymatically hydrolyzed protein sources were comparabl e with availability measures using animal methods for lysine availability. This research shows potential applications for more rapid quantitative meas urement of bioavailable lysine. (C) 2000 Elsevier Science BN. All rights re served.