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 trypt ophan-2-monooxygenase (TMO) has been developed for reagentless L-trypt ophan determination. Concentrations of L-tryptophan between 0.1 and 50 mM could be measured with the linear part of the calibration curve be tween 0.1 and 2 mM. The response time was 30s and the total analysis t ime was less than 3 min. The biosensor retained activity for greater t han 4 months, when operated daily at 25 degrees C and stored at 8 degr ees C. The biosensor was characterized by a relatively high sensitivit y to phenylalanine (54% that of L-tryptophan), a modest response to L- methionine (less than 6%) and virtually no response to other amino aci ds. However, the biosensor selectivity to L-tryptophan could be dramat ically increased when indoleacetamide (IA), a competitive inhibitor of TMO, was introduced. In the presence of 10 mu M IA, the biosensor res ponse to L-phenylalanine decreased to 74% 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. (C) 1997 Published by Elsevier Sci ence Limited.