Enzymatic characterization and polyurethane biodegradation assay of two novel esterases isolated from a polluted river

Abstract

The environmental ubiquity of plastic materials generates global concern, pollution, and health problems. Microorganisms and enzymes with plastic biodegradation potential are considered as environmentally friendly alternatives to address these issues. Interestingly, polluted environments exert selective pressure on native microbial communities that have the metabolic capacity to tolerate and transform different contaminants, including plastics. A number of enzymes have been described as polyurethane degraders. However, some of them do not possess complete characterization or efficient degradation rates. Hence, there is still a need to identify and characterize efficient enzymes for application in green processes for plastic recycling. Here, we used an environmental DNA sample isolated from the sediments of a polluted river in Mexico (Apatlaco River), which was used to construct a metagenomic fosmid library to explore the metabolic potential of microbial communities for polyurethane biodegradation. Functional screenings were performed on agar media containing the polyester polyurethane Impranil DLN (Impranil), and positively selected fosmid DNA was identified and sequenced by Illumina. Bioinformatic analyses identified two Acinetobacter genes (epux1 and epux2) encoding alpha/beta hydrolases. The genes were heterologously expressed to determine the capacity of their encoded proteins for Impranil clearing. Both Epux1 and Epux2 enzymes exhibited Impranil cleavage at 30 °C and 15 °C and ester group modifications were validated by infrared spectroscopy. Furthermore, the release of building blocks of the polymer was determined by GC-MS analysis, thus indicating their esterase/polyurethanase activity. Overall, our results demonstrate the potential of these novel bacterial enzymes for the hydrolysis of polyurethane with potential applications in the circular plastics economy.

Fig 1. E. coli clone 1–19 showing lipolytic and clearing Impranil activity.

E. coli clone 1–19 in LB agar + tributyrin and LB agar + Impranil-DLN, and negative control (E. coli Epi300/PCC2FOS-control insert) incubated for three days at 37 °C and one week at 30 °C.

Fig 2. Phylogenetic analysis and sequence alignments of Epux1 and Epux2 with other lypolitic enzymes.

A) phylogenetic analysis of Epux1 and Epux2 and 22 characterized lipolytic enzymes representing eight different families. B) Amino acid sequence alignment of Epux1 and other family IV carboxylesterases. Est8 (PDB: 4YPV), EstE5(PDB: 3FAK), and EstB (GenBank: ABY60417). Characteristic motifs for family IV are colored: oxyanion hole in cyan and pentapeptide GDSAG in yellow. C) Amino acid sequence alignment of Epux2 and other family VIII carboxylesterases. LipL (GenBank: P71778), EstA (GenBank: CAA78842), and EstIII (GenBank: AAC60471). The characteristic motif for family VIII is colored in yellow and the GXSXG motif in LipL and EstA is colored in gray. Asterisks indicate fully conserved amino acids, and amino acids that form the catalytic triad are indicated by boxes.

Fig 3. 3D structural models of Epux1 and Epux2.

A and B, ribbon representation of the folding of Epux1 and Epux2, respectively. C and D, surface representation of Epux1 and Epux2, respectively. The active site cleft in each protein is indicated by a dotted rectangle. All figures are based on AlphaFold models.

Fig 4. Enzymatic clearing activity of Epux1 and Epux2 on tributyrin and Impranil.

Enzymatic clearing assays of Epux1 and Epux2 on agar containing A) 1% tributyrin, B) 0.3% Impranil. Negative control: E.coli/BL21 pET24a cell extract.

Fig 5. Substrate specificity of Epux1 and Epux2 against p-nitrophenyl esters.

The enzymatic activity was determined at 30 °C, in KH₂PO₄ buffer (pH 7.2) using pNP-butyrate (C4), pNP-caprylate (C8), pNP-laurate (C12), pNP-palmitate (C16), or pNP-stearate (C18) as a substrate. The average values of three replicates are shown together with error bars.

Fig 6. Effect of temperature (A) and pH (B) on esterase activity of Epux1 and Epux2 with p-nitrophenyl butyrate (C4) as substrate.

The average values of three replicates are shown together with error bars.

Fig 7. Impranil clearing activity of Epux1 and Epux2 in different conditions of pH and temperature.

Reactions of 1 mL containing Impranil (1.6 mg/mL) in potassium sodium buffer (pH 7) or Britton-Robinson buffer (pH 9). Denatured Epux1 and Epux2 and Impranil suspension without enzyme were used as negative controls. Each graphic represents the average of three replicates ± standard deviation, * significant difference (α = 0.05).

Fig 8. FT-IR spectra comparison between Impranil suspension without enzymatic treatment and treatment with Epux1 (A) and Epux2 (B), and the controls with denatured Epux1 (C) or denatured Epux2 (D).

Impranil suspension without enzymatic treatment (blue), treated with Epux1 (green) and with Epux2 (magenta). As negative controls denatured Epux1 (brown) and Epux2 (clear gray) were utilized. Reactions of 1 mL; 1.6 mg/mL Impranil in Britton-Robinson buffer (pH 9), with 1.4 μM of Epux1 or Epux2, incubated 18 h at 15 °C. Each graphic represents the average of three replicates obtained by Spectragryph.

Fig 9. GC-MS chromatograms of the DCM extracts of Impranil-DLN suspensions treated with Epux1 or Epux2 and the negative control (Impranil-DLN suspension without enzymatic treatment).

Reactions of 1 mL; 1.6 mg/mL Impranil in Britton-Robinson buffer (pH 9), with 1.4 μM of Epux1 or Epux2, incubated 18 h at 15 °C.