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

Abstract

Unlike traditional recycling strategies, biodegradation is a sustainable solution for disposing of poly(ethylene terephthalate) (PET) waste. PETase, a newly identified enzyme from Ideonella sakaiensis, has high efficiency and specificity towards PET and is, thus, a prominent candidate for PET degradation. On the basis of biochemical analysis, we propose that a wide substrate-binding pocket is critical for its excellent ability to hydrolyze crystallized PET. Structure-guided site-directed mutagenesis revealed an improvement in PETase catalytic efficiency, providing valuable insight into how the molecular engineering of PETase can optimize its application in biocatalysis.

Structural studies reveal the molecular mechanism of PETase

“…The PETase-specific DS1, on the other hand, is located adjacent to the active site and connects the b7-a5 and b8-a6 loops that harbor the catalytic acid (D177) and catalytic base (H208), respectively (Fig. Interestingly, PET hydrolysis measured under reducing conditions and mutagenesis experiments indicate that DS1 depletion significantly reduces enzyme activity, suggesting that DS1 plays a critical role in PETase activity [9,[11][12][13]. In addition, the b8-a6 loop in PETase is three residues longer than that of other homologous enzymes (Fig.…”

“…Significantly, I. sakaiensis secretes a unique cutinase-like enzyme, later dubbed PETase, to hydrolyze PET to bis(2-hydroxylethyl) TPA (BHET), mono(2-hydroxyethyl) TPA (MHET), and TPA ( Fig. Here, we summarize the most recent studies of crystal structures of PETase and its complex with substrate/product analogs reported by ourselves and other groups [9][10][11][12][13]. Under physiological conditions (30°C, pH 7.0), the enzyme is 5.5-to 120-fold more efficient than previously reported PET-hydrolyzing homologs.…”

“…Recently, we and other groups independently reported the crystal structure of PETase [9][10][11][12][13]. The enzyme adopts a typical a/b-hydrolase fold, containing a twisted central b-sheet consisting of nine b-strands which is sandwiched by six a-helices ( Fig.…”

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

“…ChemSusChem 2018, 11,4018 -4025 www.chemsuschem.org Low-crystalline PET hydrolysis unders tandard reaction conditions Standard reaction conditions were determined based on our previous report. [18] As control experiments, the same volume of buffer with the enzyme solution was added to the preincubated film and incubated for 3h.T he reaction was terminated after each incubation time by adding dilution solution (500 mL, 70 %v/v MilliQ, 20 %v/v acetonitrile, and 10 %v/v formic acid). The PET hydrolysis reaction was initiated by adding PETase (final concentration 500 nm)a t3 0 8C.…”

“…[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.…”