Improved Sensitivity of a Histamine Sensor Using an Engineered Methylamine Dehydrogenase

Methylamine dehydrogenase (MADH) may be immobilized in a polypyrrole (PPy) film on an electrode surface and used as an amperometric sensor for the determination of histamine. Using site-directed mutagenesis, phenylalanine 55 on the alpha subunit of MADH was converted to alanine. This alphaF55A MADH exhibits a 400-fold lower Km value for histamine than does native MADH when assayed in solution. An alphaF55A MADH-PPy sensor was constructed, and its properties were compared to that of the native MADH-PPy sensor. The alphaF55A MADH immobilized on the electrode exhibited Michaelis-Menten behavior in response to varied concentrations of histamine with an approximately 3-fold lower Km value than that exhibited by the immobilized native MADH. The detection limit for the native MADH-PPy sensor was approximately 20 microM while the alphaF55A MADH-PPy sensor exhibited a detection limit of approximately 5 microM, a 4-fold increase compared to the native MADH-PPy sensor. This work highlights the potential value of using site-directed mutagenesis to engineer enzymes to alter and improve biosensor performance.