Decomposition of the PET Film by MHETase Using Exo-PETase Function

Abstract

Monohydroxyethyl terephthalate (MHET) hydrolase (MHE-Tase) is an enzyme known to be involved in the final degradation step of poly(ethylene terephthalate) (PET) by hydrolyzing MHET into terephthalic acid and ethylene glycol in Ideonella sakaiensis. Here, we report the extracellular production of MHETase in an active form with a proper folding. Based on the structural observations and biochemical experiments, we reveal that MHETase also functions as exo-PETase by hydrolyzing the synthesized PET pentamer. We further present that MHETase has a hydrolysis activity against the termini-generated PET film, demonstrating the exo-PETase function of the enzyme. We also develop a MHETase R411K/S416A/F424I variant with a higher BHET activity, and the variant exhibits an enhanced degradation activity against the PET film. Based on these results, we propose that MHETase plays several roles in the biodegradation of PET using the BHETase and exo-PETase activities as well as the MHET hydrolysis function.

Characterization and engineering of a two-enzyme system for plastics depolymerization

“…A similar Ca 2+ binding site was characterized for Aspergillus oryzae FaeB ( Ao FaeB), wherein it was hypothesized to have a role in stabilizing the lid domain ( 38 ). Overall, the structure of MHETase is most similar to that of FAEs, as discussed previously ( 35 , 36 ). The structural conservation between the hydrolase domains of MHETase and PETase is striking ( Fig.…”

“…The enzyme kinetics studies presented here reveal a substantial reduction in activity for the S131G, E226T, and F495I MHETase mutants, indicating that these positions play important roles in substrate specificity and catalytic efficiency. A previous study also demonstrated greatly reduced hydrolytic activity by a F415S variant ( 36 ). Additionally, two homologs identified via bioinformatics analysis from C. thiooxydans and Hydrogenophaga sp.…”

“…In biological systems, complete depolymerization to monomers can be necessary for microbial uptake and growth, as in I. sakaiensis wherein MHETase is the enzymatic partner to PETase, together allowing for the complete degradation of PET to TPA and EG for catabolism ( 10 ). Prior studies presenting MHETase crystal structures focused upon understanding and tuning substrate specificity, particularly the rational engineering of MHETase to impart BHET hydrolysis activity ( 35 , 36 ). Drawing inspiration from our structural analyses, this complementary study offers further insights into the two-enzyme PETase/MHETase system.…”

“…Conversely, the structure and function of the MHETase enzyme is far less characterized, with only two published studies focused on MHETase structure and engineering to date ( 35 , 36 ). These studies report structures at 2.1 to 2.2 Å resolution, wherein the similarity to ferulic acid esterase (FAE) is noted ( 37 , 38 ).…”

An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films

“…Without immediate interventions in plastic usage and industrial-scale changes to the intended life cycle of plastics, plastic consumption is projected to grow 300% between 2014 and 2050 3 . To address this exponentially growing crisis, researchers have begun to turn attention towards enzymebased strategies for recycling and bioremediation [4][5][6][7][8][9] . Careful kinetic studies must be performed to identify accurate structure-function relationships for plastic-hydrolase engineering, but analyses of the initial phases of the reactions are not commonly reported, sometimes leading to misinterpretation of effects of mutations.…”