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.

Figure 1

Concomitant engineering of two domains of Cbotu_EstA (PDB-Code 5AH1) to improve hydrolysis of PET. (I) The truncation of the N-terminal extra domain (red arrow) of 71 amino acids (marked in red) should lead to improved dynamics of the lid structure and the display of a hydrophobic patch for a better adsorption to hydrophobic substrates. (II) The substitution of residues (orange sticks) present in the zinc-binding domain (blue arrow) is expected to increase the activity of the enzyme on bulky water-insoluble substrates. The active site (Ser-182, His-426, Asp-384) is highlighted with a blue circle and the zinc ion in the zinc-binding domain in magenta. The representation was created using YASARA view (v. 14.7.17).

Figure 2

SDS-PAGE analysis (4–12%) of Cbotu_EstA wild-type and variants expressed in E. coli BL21-Gold(DE3) with 0.05 mM IPTG and purified by IMAC. Lane 1: Cbotu_EstA; lane 2: del71Cbotu_EstA; lane 3: del71Cbotu_EstA_S127A; lane 4: del71Cbotu_EstA_W129A; lane 5: del71Cbotu_EstA_F154Y; lane 6: del71Cbotu_EstA_S199A; lane 7: del71Cbotu_EstA_W274H; lane M: pre-stained protein molecular ladder Protein Marker IV (Peqlab, Germany). Image cropped and full-length gel found in Supplementary Information.

Figure 3

Michaelis-Menten plot of Cbotu_EstA, del71Cbotu_EstA, del71Cbotu_EstA_W129A, and del71Cbotu_EstA_S199A with para-nitrophenyl butyrate (pNPB) in a concentration range 0.3–15 mM. Michaelis-Menten plot of all the variants can be found in Supplementary Information (Fig. S1).

Figure 4

Thermal stability of Cbotu_EstA wild-type and variants determined with the soluble substrate p-NPB. The columns represent the residual activities of the purified enzymes after 24 h incubation at 50 °C.

Figure 5

Enzymatic hydrolysis of PET by Cbotu_EstA wild-type and variants. (a) Possible PET hydrolysis pathway. The hydrolysis products Ta and MHET were detected by HPLC analysis, while the chromatogram was acquired at 241 nm. The grey arrow shows the possible cleavage site hydrolyzed by the Cbout_EstA variants. (b) PET hydrolysis of Cbotu_EstA wild-type and variants. The data are the mean value of three different measurements and the bars represent the standard deviation.