Response to Comment on “A bacterium that degrades and assimilates poly(ethylene terephthalate)”

Abstract

Yang et al. suggest that the use of low-crystallinity poly(ethylene terephthalate) (PET) exaggerates our results. However, the primary focus of our study was identifying an organism capable of the biological degradation and assimilation of PET, regardless of its crystallinity. We provide additional PET depolymerization data that further support several other lines of data showing PET assimilation by growing cells of Ideonella sakaiensis. W e appreciate the Comment by Yang et al.(1) and are grateful for this opportunity to explain the context of how our work builds on previous studies. The intent of our study was to isolate and describe a microorganism that can degrade and assimilate poly(ethylene terephthalate) (PET) (2). Therefore, we only cited the pioneering works that report specific microorganisms able to grow on PET (3, 4). In a previous study, Sharon and Sharon (5) confirmed microbial PET degradation by semiisolated microorganisms, where the involvement of Nocardia species was implied by microscopic observation. However, the contribution of other microorganisms was not ruled out, nor was the possibility that the PET was degraded by the resting cells harboring PET-hydrolyzable enzymes. We identified the PET hydrolase (PETase) gene based on amino acid sequence identity with a known hydrolase from Thermobifida fusca (TfH) that exhibited PET-hydrolyzing activity (6), similar to other PET-hydrolyzing enzymes (7-10). We further referred cutinase from Humicola insolens (HiC) (11) and seven other enzymes in the supplementary materials [table S2 in (2)]. The functions of these enzymes, particularly TfH, LCC (9), and FsC (10), greatly contributed to the understanding of PETase enzymology.With the intention of isolating a specific microorganism that can use PET for growth, even if the PET is mostly in the amorphous form, the extent of crystallinity is of secondary importance. We described the motivation for using low-crystallinity (1.9%) PET and the observation that the structure of crystallized PET hampered the enzymatic hydrolysis of its ester linkages (8, 12) in our Report.Based on the proton nuclear magnetic resonance and gel permeation chromatography profiles of the degraded PET film, we concluded that the degradation by the consortium termed "no. 46" proceeded from the PET surface (2). To support this inference, we analyzed the surface of the PET film using x-ray photoelectron spectroscopy (XPS). The XPS spectrum for degraded PET film by no. 46 showed that a new peak appeared at 0.6 eV higher in binding energy than that observed for the O 1s peak of C-O-C linkage (532.5 eV), indicating the formation of the C-O-H linkage by hydrolysis of the ester linkage, which should either be of hydroxyl or carboxyl groups. To accurately determine the surface functional groups, each group was labeled with heptafluorobutyryl chloride (for -OH) and 1,1-carbonyldiimidazole (for -COOH) (13) and analyzed by XPS. Consequently, the peaks of F 1S (687.5 eV) and N 1S (398.5 eV) after degradation dramatically increased (Fig. 1A). The ...

Protein Crystallography and Site‐Direct Mutagenesis Analysis of the Poly(ethylene terephthalate) Hydrolase PETase from Ideonella sakaiensis

“…For instance, the preference of this system for p ‐nitrophenol (pNP)‐linked aliphatic esters is lower than that of TfH, LCC, and FsC, but in terms of PET films, the ability of the PETase/MHETase system is 120, 5.5, and 88 times higher than that of TfH, LCC, and FsC, respectively . Essentially, PETase shows better performance on highly crystallized PET, commonly regarded as a knotty polymer for hydrolysis, than other enzymes …”

Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation

“…Recently, a new bacterial species, Ideonella sakaiensis , which can use PET as a carbon source, was isolated 17 – 20 . The PETase of I. sakaiensis ( Is PETase) can degrade PET at a moderate temperature (30 °C) and has relatively higher activity than other PET-degrading enzymes, such as cutinases and lipases 17 .…”

Acceleration of Enzymatic Degradation of Poly(ethylene terephthalate) by Surface Coating with Anionic Surfactants

“…[4][5][6][7][8][9][10][11] For example, cutinases from Thermobifida fusca [8,9] and the most efficient PET hydrolyzing esterase, leaf and branch compost cutinase( LCC). [12][13][14][15][16][17][18][19][20] Although PETase is highly specific to PET,t he enzymes hows poor catalytic activity and is difficult to use in industrial processes. [12][13][14][15][16][17][18][19][20] Although PETase is highly specific to PET,t he enzymes hows poor catalytic activity and is difficult to use in industrial processes.…”

“…[10,11] In 2016, our research group also identified aP ET-specific hydrolase (PETase, EC: 3.1.1.101, PDBID:5 XJH) from the PET assimilating bacterium, Ideonella sakaiensis (Figure1b). [12][13][14][15][16][17][18][19][20] Although PETase is highly specific to PET,t he enzymes hows poor catalytic activity and is difficult to use in industrial processes. [12] In 2013, Espino-Rammer et al reported an interesting approach to accelerate the catalytic activityo fP ET hydrolyzing enzyme Humicola insolens cutinase (HiC) by using additives.…”