A biosensor for all D-amino acids using evolved D-amino acid oxidase.
J Biotechnol
; 135(4): 377-84, 2008 Jul 31.
Article
en En
| MEDLINE
| ID: mdl-18588925
ABSTRACT
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Rhodotorula
/
Técnicas Biosensibles
/
D-Aminoácido Oxidasa
/
Aminoácidos
Idioma:
En
Revista:
J Biotechnol
Asunto de la revista:
BIOTECNOLOGIA
Año:
2008
Tipo del documento:
Article
País de afiliación:
Italia