Continuous-flow step gradient mass spectrometry based method for the determination of kinetic parameters of immobilized mushroom tyrosinase in equilibrating conditions: comparison with free enzyme.

A mass spectrometry (MS)-based methodology for enzymatic assay in equilibrium conditions was designed and evaluated. This on-line assay involves the introduction of a continuous-flow step gradient (CFSG) of a substrate solution in the column containing immobilized enzyme and the simultaneous tracking of the product formation. We showed that the constant concentration of substrate in the entire bioreactor for an appropriate duration ensures the equilibration of the studied enzyme (mushroom tyrosinase). Under these conditions, it was demonstrated also that the kinetic and enzymatic parameters (Michaelis-Menten constant, K(M) , the maximal specific activity, SA(max)) are independent of the flow rate of the mobile phase. The feasibility of the mentioned approach for inhibitory tests was also investigated. The coupling of the mass spectrometer to the bio-reactor allows the selective monitoring of the enzymatic reaction products and increases their detection level. Very high sensitivity, 500 pmol/min/column, and selective monitoring of the products of the enzymatic reaction are allowed by MS detection. The methodology developed here constitutes a sensitive analytical tool to study enzymes requiring long equilibration times.