title: Improved sensitivity of a histamine sensor using an engineered methylamine dehydrogenase.
abstract: 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.
authors.affiliation: Department of Chemistry, Jackson State University, Mississippi 39217, USA.
authors.forename: Lili
authors.initials: L
authors.lastname: Bao
authors.forename: Dapeng
authors.initials: D
authors.lastname: Sun
authors.forename: Hiroyasu
authors.initials: H
authors.lastname: Tachikawa
authors.forename: Victor L
authors.initials: VL
authors.lastname: Davidson
mesh_terms: D015374:Biosensing Techniques; D004800:Enzymes, Immobilized; D006632:Histamine / Q000032:analysis*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering
chemical_list: D004800:Enzymes, Immobilized; D006632:Histamine; C021567:methylamine dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors