Biundo_2017_Catal.Sci.Technol_7_1440

The carboxylesterase from Clostridium botulinum (Cbotu_EstA) has been shown to hydrolyze the surface of the polyester poly.butylene adipate-co-terephthalate) (PBAT) releasing the monomeric building blocks. Cbotu_EstA contains a zinc ion, tetrahedrally coordinated by two histidine and two aspartic acid residues, which is buried inside an extra domain typical for members of the I. 5 lipase family. To elucidate the role of this extra domain with regard to polyester hydrolysis, variants of the zinc-binding domain were constructed and expressed in E. coli BL21-Gold.DE3). These enzyme variants were characterized with respect to their specific activity, kinetic parameters and thermostability on soluble substrates as well as on PBAT. All the variants exhibited a similar affinity towards the small substrate para-nitrophenyl butyrate (pNPB), with KM values between 0.4 and 1.2 mM, while the catalytic efficiency decreased approximately 1000-fold for the zinc-binding variants and 5-fold for the zinc cavity variants. Moreover, all four variants of the zinccoordination site (D130L, H150F, H156F, and D302L) showed a loss of thermostability. However, H156F and D302L revealed a drastic loss of thermostability compared to D130L and H150F. Nevertheless, compared to Cbotu_EstA, variants carrying substitutions of amino acids in the zinc-binding domain were able to release up to 10 times more soluble products from the polymeric substrate PBAT. The thermostability at 50 degrees C was increased in the case of F154Y and W274H, carrying more hydrophilic residues. These data clearly demonstrate the importance of different regions of the zinc-binding domain for the hydrolysis of polyesters like PBAT.