The suitability of several chromogenic substrates to detect Alcalase (Novo) and other protease enzymes in sensing devices was evaluated. The sensitivity of the substrates was evaluated from their K(cat) values and their stability to spontaneous breakdown was determined over an 8-h period. p-Nitrophenyl trimethylacetate showed reasonable sensitivity but was the least stable of the substrates. The other substrates were based on p-nitroanalide chromophores and were very stable. The substrate which showed the highest sensitivity to Alcalase was N-succinyl-L-alanyl-L-alanyl-L-ananyl-p-nitroanilide (k3= 50.25 μmol AU-1min-1). This substrate was selected for an 8-h assay. Esperase (Novo) and Subtilisin Carlsberg were compared with Alcalase with the substrate p-nitrophenyl trimethylacetate. A further evaluation of N-succinyl-L-alanyl-L-alanyl-L-alanyl-p-nitroanilide was made by incubating this substrate with Alcalase over an 8-h period. There was a gradual reduction in the rate over the 8-h period but it is suggested that the enzyme-substrate reaction is sufficiently stable for incorporation in a sensor for long-term monitoring.
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