Fashioned enzymes: adjustment of properties of bacterial alkaline phosphatase to the enzymatic labelling

Enzymatic tracers can be easily designed by genetic fusion between a gene encoding a protein or a protein domain and the gene of the E coli alkaline phosphatase. With the aim to enhance the sensitivity of the tests developed using such conjugates, we describe a strategy to improve the bacterial alkaline phosphatase efficacy. In a first step we generated an inactive form of the enzyme and in a second step we looked for revertants. Finally, the introduction of only two mutations provided an enzyme with a catalytic activity increased 40-fold relative to that of the wild-type enzyme while maintaining high thermostability. A large panel of 44 lab tests > can be quickly designed using the modified enzyme and the gene fusion approach, with a similar sensitivity to which obtained with conventional tracers.