Development of a D-alanine sensor for the monitoring of a fermentation using the improved selectivity by the combination of D-amino acid oxidase and pyruvate oxidase.
A D-alanine (D-Ala) sensor for the monitoring of a fermentation process was developed using flow injection analysis (FIA). The FIA system consisted of a D-amino acid oxidase (D-AAOx) reactor, a Pyruvate oxidase (PyOx) electrode and a contrast electrode in the flow cell, and through the oxidation of D-amino acids in the D-AAOx reactor, pyruvic acid was formed only from D-Ala. The pyruvic acid was further oxidized with PyOx via the D-AAOx reaction. The amount of oxygen consumed in the PyOx reaction was proportional to the amount of D-Ala. It was possible to continuously repeat the assay up to 60 times at pH 6.8 and a flow rate of 0.18-ml min(-1). A linear relationship was obtained in the range of 0.1-1 mM D-Ala with a correlation coefficient of 0.987 and the detection limit was 0.05 mM. The relative standard deviation (R.S.D.) was 4.9% (n=5) for 0.5 mM D-Ala. The D-Ala content in some fish sauces was also determined using the proposed sensor system. The results obtained indicated a linear relationship between the amounts of D-Ala determined by the proposed sensor system and the conventional method. From the results, even if the substrate specificity of the enzyme (D-AAOx) was low, it was evident that the concentration of the original material (D-Ala) could be determined specifically when the first reaction product was changed by the second reaction (PyOx).
Inaba
Yohei
Y
Department of Food Science and Technology, Tokyo University of Fisheries, 5-7 Kounan 4, Minato-ku, Tokyo 108-8477, Japan.
Mizukami
Kentaro
K
Hamada-Sato
Naoko
N
Kobayashi
Takeshi
T
Imada
Chiaki
C
Watanabe
Etsuo
E
D000409:Alanine / Q000032:analysis* / Q000737:chemistry*; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods; D003605:D-Amino-Acid Oxidase / Q000737:chemistry*; D004563:Electrochemistry / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D017022:Flow Injection Analysis / Q000295:instrumentation* / Q000379:methods; D009862:Online Systems; D011771:Pyruvate Oxidase / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D011771:Pyruvate Oxidase; D003605:D-Amino-Acid Oxidase; D000409:Alanine