A tryptophan-2-monooxygenase based amperometric biosensor for L-tryptophan determination: use of a competitive inhibitor as a tool for selectivity increase.
A new flow-injection amperometric biosensor based on immobilized tryptophan-2-monooxygenase (TMO) has been developed for reagentless L-tryptophan determination. Concentrations of L-tryptophan between 0.1 and 50 mM could be measured with the linear part of the calibration curve between 0.1 and 2 mM. The response time was 30 s and the total analysis time was less than 3 min. The biosensor retained activity for greater than 4 months, when operated daily at 25 degrees C and stored at 8 degrees C. The biosensor was characterized by a relatively high sensitivity to phenylalanine (54% that of L-tryptophan), a modest response to L-methionine (less than 6%) and virtually no response to other amino acids. However, the biosensor selectivity to L-tryptophan could be dramatically increased when indoleacetamide (IA), a competitive inhibitor of TMO, was introduced. In the presence of 10 microM IA, the biosensor response to L-phenylalanine decreased to 7-4% of the unaffected rate for L-tryptophan. In the absence of L-tryptophan and IA the biosensor could be used for L-phenylalanine determination in the concentration range from 1 to 50 mM. The biosensor was successfully used for L-tryptophan determination in nutritional broth.
Simonian
A L
AL
Texas A&M University, Department of Biochemistry and Biophysics, College Station 77843, USA.
Rainina
E I
EI
Fitzpatrick
P F
PF
Wild
J R
JR
D015374:Biosensing Techniques; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D010649:Phenylalanine / Q000032:analysis; D012680:Sensitivity and Specificity; D014364:Tryptophan / Q000032:analysis*
D010649:Phenylalanine; D014364:Tryptophan; D006899:Mixed Function Oxygenases; C023204:tryptophan 2-monooxygenase