PACKTEST for L-Glutamate Quantification: Development of On-site and High-throughput Analytical Kits Using L-Glutamate Oxidase Mutant

L-glutamate is an umami component found in various foods, and L-glutamate oxidase (LGOX, EC 1.4.3.11) is a FAD-dependent oxidoreductase that catalyzes the oxidative deamination of L-glutamate. A recently reported single-chain LGOX mutant from Streptomyces sp. X-119-6 is a potential material for L-glutamate sensors because this mutant is easier to prepare than the wild type. Herein, we report the development of a simple analytical kit using this LGOX mutant for the on-site quantification of L-glutamate. The kit named PACKTEST was developed using a colorimetric method to estimate the L-glutamate concentration in the range of 1-50 mg/L through the visual comparison of the resulting sample color with a standard color sequence. In addition, the PACKTEST kit was combined with a portable photometer for the spectrophotometric quantification of L-glutamate in the range of 0.5-12.0 mg/L. It was used to quantify L-glutamate in commercially available soy sauces, and the results were in good agreement (r r = 0.97) with the values obtained by post-column derivatized ion-exchange chromatography. Furthermore, we loaded and dried all the reagents, including enzymes, on a 96- well plate to prepare an enzyme-based sensing device, which showed potential for the high- throughput quantification of L-glutamate.