A dual enzyme system composed of a polyester hydrolase and a carboxylesterase enhances the biocatalytic degradation of polyethylene terephthalate films

Abstract

TfCut2 from Thermobifida fusca KW3 and the metagenome-derived LC-cutinase are bacterial polyester hydrolases capable of efficiently degrading polyethylene terephthalate (PET) films. Since the enzymatic PET hydrolysis is inhibited by the degradation intermediate mono-(2-hydroxyethyl) terephthalate (MHET), a dual enzyme system consisting of a polyester hydrolase and the immobilized carboxylesterase TfCa from Thermobifida fusca KW3 was employed for the hydrolysis of PET films at 60°C. HPLC analysis of the reaction products obtained after 24 h of hydrolysis showed an increased amount of soluble products with a lower proportion of MHET in the presence of the immobilized TfCa. The results indicated a continuous hydrolysis of the inhibitory MHET by the immobilized TfCa and demonstrated its advantage as a second biocatalyst in combination with a polyester hydrolase for an efficient degradation oft PET films. The dual enzyme system with LC-cutinase produced a 2.4-fold higher amount of degradation products compared to TfCut2 after a reaction time of 24 h confirming the superior activity of his polyester hydrolase against PET films.

Degradation and metabolism of synthetic plastics and associated products byPseudomonassp.: capabilities and challenges

“…; Barth et al . ). For instance, the MHET by‐product of PET hydrolysis is inhibitory of enzymes found in Thermobifida fusca KW3, and glyoxylic acid inhibits Flavobacterium sp.…”

“…during PEG degradation (Gu ; Barth et al . ). Potential inhibitory effects of plastic degradation by‐products on Pseudomonas species remain to be fully understood.…”

Screening of commercial enzymes for poly(ethylene terephthalate) (PET) hydrolysis and synergy studies on different substrate sources

“…Thus, the stabilization of MHET concentration from 10 to 14 days of reaction may suggest that this is a limiting condition for MHET accumulation; however, systematic enzyme kinetic studies are needed to support the hypothesis of inhibition by MHET, as done by Barth et al [2]. At 14 days of reaction, TPA was the main product of the reactions in the seven substrates (χ TPA = 0.552-0.938), with the only exception being when HiC was used as the only biocatalyst for amorphous PET hydrolysis (χ TPA and χ MHET were 0.494 and 0.495, respectively).…”

“…The simultaneous use of two enzymes for PET film hydrolysis was investigated by Barth et al [2]. Authors used a T. fusca carboxylesterase (TfCa) to complement the good PET depolymerizing activity of either a T. fusca cutinase or a metagenome-derived LC-cutinase, and observed that the higher the amount of TfCa, the higher the proportion of TPA over MHET.…”

“…For example, Yoshida et al [32] identified two different catalytic properties from Ideonella sakaiensis enzymes, defining them as PETase and MHETase. Also, Barth et al [2] reported a dual enzyme reaction system consisted with a polyester hydrolase, LCcutinase, hydrolyzing PET film chains and an immobilized carboxylesterase, TfCa KW3 from Thermobida fusca, as the second enzyme applied to eliminate inhibitory intermediates produced from PET hydrolysis. Similar properties were observed by Carniel et al [8], who demonstrated that the Humicola insolens cutinase and the Candida antarctica lipase B act synergistically to convert PET into its final monomers.…”