The implementation of biocatalytic procedures in flow systems as an alternative to conventional batch assays minimizes the factors more important that affect to enzyme (pH, ionic strength and temperature) allowing the maximization of enzyme activity. An important aspect for automation of enzyme based assays, as waste of enzyme solutions/suspensions is minimized because well defined portions are applied in sequential injection analysis (SIA).A SIA method to evaluate the enzymatic activity of �-glucosidase in solution was development. For the activity study 4-Nitrophenyl-�-D-glucopiranoside (pNPG) was used as substrate; the reaction product (p-nitrophenol) was measured at 400 nm. A first factorial design 25-1 plus three central points was used to evaluate volume of substrate, volume of enzyme, flow-rate, pH and temperature. A second factorial design 22 plus three central points was also used to study the behaviour of temperature and pH over the enzymatic activity. The optimal conditions were: pH 4 for the enzymatic reaction and pH 10 for the measurement of p-nitrophenol, flow-rate of 20 �L s-1, 50 �L of of pNPG at different concentrations  and 10 �L of the enzyme solution (1 unit mL-1). The kinetics parameters found were: Km= (7.22�0.08) mM y Vmax = (0.00697�0.00003) mM s-1, y kcat = (1.063�0.005) s-1.  The results also showed that the enzyme could work at different temperatures with high activity from 20 �C to 70 �C and at 80 �C had activity yet.

Evaluation of the activity of �-glucosidase in solution by sequential injection analysis

248th National Meeting (2014)

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