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Cristina G. Silvestre & Maria Cristina R. Ramos
A possible undergraduate microscale experiment for enzymology is presented. In this study, the ability of Serratia marcescens to hydrolyze gelatin is exploited and the amount of hydrolyzed gelatin was measured using the Bradford Assay.Briefly, to study the enzymatic activity of S. marcescens secreted-gelatinase, gelatin solution was incubated with an 18th hour supernatant of S. marcescens. After incubation, the amount of hydrolyzed gelatin was monitored. The optimal activity of the enzyme was observed at 1 h incubation time, 1.25% (w/v) gelatin as the substrate, 37°C, pH 6.7 and ZnCl2 as activator. For the kinetic properties using gelatin as the substrate, the vmax and KM of the gelatinase are 0.23 (mg/mL) min–1 and 10.33 mg/mL, respectively, using the Lineweaver-Burk plot. The inhibitory effects of EDTA and citric acid were also studied. When gelatin is incubated with S. marcescens supernatant in the presence of EDTA or citrate, the slopes of the Lineweaver-Burk plots were not altered, and the KM and vmax values of the inhibited reactions decreased. This behavior indicates that these substances are uncompetitive inhibitors. A summarizedprotocol which can be performed in a 3 h laboratory period when the bacterial supernatant is prepared beforehand is presented in this study. This protocol enables students to learn quantitative determination of protein using Bradford assay at the same time.
Keywords: matrix metalloproteinases, gelatinase, enzyme kinetics and inhibition,
Lineweaver Burk plot
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