Synergistic effect of mutagenesis and truncation to improve a polyesterase from Clostridium botulinum for polyester hydrolysis

Abstract

The activity of the esterase (Cbotu_EstA) from Clostridium botulinum on the polyester poly(ethylene terephthalate) (PET) was improved by concomitant engineering of two different domains. On the one hand, the zinc-binding domain present in Cbotu_EstA was subjected to site-directed mutagenesis. On the other hand, a specific domain consisting of 71 amino acids at the N-terminus of the enzyme was deleted. Interestingly, a combination of substitution of residues present in the zinc-binding domain (e.g. S199A) synergistically increased the activity of the enzyme on PET seven fold when combined to the truncation of 71 amino acids at the N-terminus of the enzyme only. Overall, when compared to the native enzyme, the combination of truncation and substitutions in the zinc-binding domain lead to a 50-fold activity improvement. Moreover, analysis of the kinetic parameters of the Cbotu_EstA variants indicated a clear shift of activity from water soluble (i.e. para-nitrophenyl butyrate) to insoluble polymeric substrates. These results evidently show that the interaction with non-natural polymeric substrates provides targets for enzyme engineering.

Current knowledge on enzymatic PET degradation and its possible application to waste stream management and other fields

“…Perz et al (2016) cloned two genes coding for the esterases Cbotu_EstA and Cbotu_EstB from an anaerobic bacterium, Clostridium botulinum ATCC 3502, and analyzed the crystal structure of the more efficient enzyme Cbotu_EstA. Cbotu_EstA could hydrolyze PET at 50 °C only by a marginal level, but truncation of the N-terminal 71 residues exposed a hydrophobic patch on the surface, similar to a cutinase, and introduction of mutation in the zinc-binding domain improved the hydrolytic activity on PET at 50 °C (Biundo et al 2018a). Based on the complete genome sequence of Thermomonospora curvata -type strain (B9) (Chertkov et al 2011), Tcur1278 and Tcur0390 have been cloned from T. curvata DSM43183 (Wei et al 2014b).…”

“…The roles of Ca 2+ and Mg 2+ in Thc_Cut2 have not been elucidated yet, but they are most probably the same as those in TfCut2. Cbotu_EstA has a zinc-binding domain, and mutation of amino acids in this domain increases the activity on PET without changing the zinc-binding ability (Biundo et al 2018a). The role of this zinc-binding domain remains to be elucidated but would appear to be different from those of metal-binding domains in cutinases.…”

Current Advances in the Biodegradation and Bioconversion of Polyethylene Terephthalate

“…The hydrolases, including lipases [31][32][33][34], cutinases [35][36][37][38][39][40][41][42], esterases [43][44][45][46], PETase [12] and MHETase [12], that can degrade PET have been identified. Among them, lipases have the lowest hydrolysis activity of PET mainly because their catalytic centers are covered by lid structures, which limits the hydrolases' contact and catalysis with the substrate PET.…”

Potential Use of Microbial Enzymes for the Conversion of Plastic Waste Into Value-Added Products: A Viable Solution

“…The hydrolysis in the second step using the HiC yielding 97% pure TPA, which was comparable with commercial synthetic grade TPA (98%). Biundo et al (2018) improved the activity of an esterase (Cbotu_EstA) from Clostridium botulinum on PET by site-directed mutations in zinc-binding domain and deleted the domain consisting of 71 amino acids at the N-terminus. The zinc-binding domain of the native enzyme had 50-fold lower activity than the combination with truncations and substitution.…”