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

Abstract

Enzymatic hydrolysis of polyethylene terephthalate (PET) has been the subject of extensive previous research that can be grouped into two categories, viz. enzymatic surface modification of polyester fibers and management of PET waste by enzymatic hydrolysis. Different enzymes with rather specific properties are required for these two processes. Enzymatic surface modification is possible with several hydrolases, such as lipases, carboxylesterases, cutinases, and proteases. These enzymes should be designated as PET surface-modifying enzymes and should not degrade the building blocks of PET but should hydrolyze the surface polymer chain so that the intensity of PET is not weakened. Conversely, management of PET waste requires substantial degradation of the building blocks of PET; therefore, only a limited number of cutinases have been recognized as PET hydrolases since the first PET hydrolase was discovered by Müller et al. (Macromol Rapid Commun 26:1400-1405, 2005). Here, we introduce current knowledge on enzymatic degradation of PET with a focus on the key class of enzymes, PET hydrolases, pertaining to the definition of enzymatic requirements for PET hydrolysis, structural analyses of PET hydrolases, and the reaction mechanisms. This review gives a deep insight into the structural basis and dynamics of PET hydrolases based on the recent progress in X-ray crystallography. Based on the knowledge accumulated to date, we discuss the potential for PET hydrolysis applications, such as in designing waste stream management.

Keywords: Catalytic mechanism; Cutinase; PET hydrolase; PETase; Potential application; Structural analyses.

Fig. 1

Image of PET film and its relevance to enzymatic attack. a PET film structure and roles of PET-modifying enzyme and PET hydrolase. Partially reproduced from Oda et al. (2018). Copyright 2018 Springer. b Preferential attack on amorphous region of PET film. BHET, bis(hydroxyethyl)terephthalate; MHET, mono(hydroxyethyl)terephthalate; TPA, terephthalic acid

Fig. 2

Phylogenetic tree for amino acid sequences of the reported PET hydrolases and their homologs. A multiple alignment and a tree were constructed using the program ClustalW2 (Larkin et al. 2007). The tree was generated by the neighbor-joining algorithm using sites without any gaps and was displayed as a midpoint-rooted tree using the program Dendroscape 3 (Huson and Scornavacca 2012). Numbers on the ancestor nodes are bootstrap values calculated by 1000 bootstrap samples. Actinomycete cutinases are represented using the enzyme names shown in Table 1. Six additional homologs for PETase were taken from the UniProt/TrEMBL database. GenBank accession AB066349 is poly(tetramethylene succinate) depolymerase from Acidovorax delafieldii (Uchida et al. 2002). The other five accession IDs are uncharacterized proteins found in bacteria as follows: ARN19491.1 and ARN19002.1 are from Rhizobacter gummiphilus, OGB27210.1 and OGB26481.1 are from Burkholderiales bacterium, and AKJ29164.1 is from Polyangium brachysporum. Two remote bacterial homologs (ADK73612.1 and CAA37220.1) were included to determine the root. ADK73612.1 is cutinase A from Pseudomonas pseudoalcaligenes, and CAA37220.1 is lipase 1 from Moraxella sp. (strain TA144)

Fig. 3

Comparison of overall 3D structures of Cut190* and PETase. The main chains are shown in ribbon models, the side chains of catalytic triads are shown in ball and stick models, and Ca²⁺ binding sites and SS bonds are shown in stick models. Substrates are shown in space-filling models. Note that serine in the catalytic triad (S176 or S131) is replaced with alanine. a Cut190 (PDB ID: 5ZNO), which has three Ca²⁺ binding sites and one SS bond. The bound substrate (ethyl succinate) is taken from PDB ID: 5ZRR. b PETase (PDB ID: 5XH3), which has no Ca²⁺ binding site and two SS bonds. The bound substrate is 1-(2-hydroxyethyl) 4-methyl terephthalate. Molecular graphics were generated using UCSF Chimera (Pettersen et al. 2004)