A biosensor for all D-amino acids using evolved D-amino acid oxidase.
Determination of the D-amino acid content in foods and in biological samples is a very important task. In order to achieve this goal we developed a biosensor employing the flavoenzyme D-amino acid oxidase from the yeast Rhodotorula gracilis. To produce a device in which the D-amino acid composition does not alter the results, both the wild-type and a number of mutants obtained by rational design and directed evolution approaches were used. An analysis of D-amino acid oxidase mutants activity on D-amino acid mixtures containing various ratios of neutral, acidic, and basic substrates identified the Amberzyme-immobilized T60A/Q144R/K152E and M213G mutants as the best choice: their response shows an only limited dependence on the solution composition when at least 20% of the D-amino acid is made up of D-alanine (standard error is approximately 5-9%). This is the first report, to our knowledge, demonstrating that the entire D-amino acid content can be determined by using a screen-printed electrode amperometric biosensor, with a detection limit of 0.25 mM and a mean response time of 10-15 min. The D-amino acid assay based on R. gracilis DAAO-biosensor is inexpensive, simple to perform, and rapid: the D-amino acid concentration of a variety of biological samples can be investigated using this assay.
Rosini
Elena
E
Dipartimento di Biotecnologie e Scienze Molecolari, Università degli Studi dell'Insubria, via J.H. Dunant 3, 21100 Varese, Italy.
Molla
Gianluca
G
Rossetti
Carlo
C
Pilone
Mirella S
MS
Pollegioni
Loredano
L
Sacchi
Silvia
S
D000596:Amino Acids / Q000032:analysis*; D015374:Biosensing Techniques / Q000295:instrumentation*; D003605:D-Amino-Acid Oxidase / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D012248:Rhodotorula / Q000201:enzymology*; D013379:Substrate Specificity
D000596:Amino Acids; D004800:Enzymes, Immobilized; D050505:Mutant Proteins; D003605:D-Amino-Acid Oxidase