On the substrate preference of glutaryl acylases

The substrate preferences of three acylases – two wild-type enzymes and an evolved variant obtained by directed evolution – which are prototypical enzymes for glutaryl-7-ACA acylase and cephalosporin C acylase subfamilies, have been investigated. A preliminary screening of enzymes’ performances on a large set of substrates has been carried out by a colorimetric assay performed in 96-well plates and by a pH-Stat monitoring the hydrolytic activities. Subsequently, kinetic data for selected substrates have been determined, thus elucidating the substrate preference of members of glutaryl-7-ACA acylase vs. cephalosporin C acylase subfamilies. These achievements pave the way to the ability of choosing the best enzyme for the hydrolysis of different compounds of industrial importance.

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► The activity of three glutaryl acylases on a number of compounds has been tested.
► We used two wild-type enzymes and an evolved variant obtained by directed evolution.
► These are prototypical enzymes for Gl-7-ACA acylase and CephC acylase subfamilies.
► The kinetic parameters for selected cephalosporin derivatives have been determined.
► The substrate preference of Gl-7-ACA vs. CephC acylase subfamilies has been elucidated.