Monitoring of alpha-ketoglutarate in a fermentation process using expandedbed enzyme reactors

A bienzyme flow injection system is presented for the monitoring of alpha - ketoglutarate produced in a fermentation process, using glutamate dehydroge nase (GDH) and glutamate oxidase (GlOx) immobilised in two serially connect ed expanded bed reactors. The use of expanded bed resulted in unhindered pa ssage of the bacterial cells through the columns, and thereby the need of a separate filtering step (e.g. microdialysis) was avoided. In the first rea ctor, a-ketoglutarate was converted to L-glutamate by GDH in the presence o f ammonia and NADH. In the following reactor, L-glutamate was converted by GlOx to alpha -ketoglutarate, ammonia and hydrogen peroxide, which was dete cted in an electrochemical flow-through cell at +650 mV vs. Pt/(0.1 M KCl). The detection limit of alpha -ketoglutarate in the coupled packed bed reac tors was 1 muM (defined as 3 S/N), the linear range 0-100 muM, and the sens itivity 0.80 nA/muM (R-2 0.99). In the coupled expanded bed reactors, the d etection limit of alpha -ketoglutarate was 7 muM (defined as 3 S/N), the li near range and the sensitivity being 0-500 muM and 0.11 nA/muM (R-2 1.00), respectively. The response time (defined as the time between peak rise and return to baseline) was 5 min for coupled packed beds (injection of superna tant), and 12 min for coupled expanded beds (injection of sample containing cellular and particulate matter). Several other parameters, such as reacto r stability, flow rate dependency, bed expansion, glutamate interference, e tc. were investigated and characterised. When analysing real samples from a fermentation broth, the same results were obtained independent of the natu re of the reactor system (packed or expanded bed). The hereby described sys tem can easily be automatised and controlled from a personal computer. (C) 2001 Elsevier Science B.V. All rights reserved.