Cutinases are enzymes known to degrade polyester‐type plastics. Est119, a plastic‐degrading type of cutinase from Thermobifida alba AHK119 (herein called Ta_cut), shows a broad substrate specificity toward polyesters, and can degrade substrates including polylactic acid (PLA). However, the PLA‐degrading mechanism of cutinases is still poorly understood. Here, we report the structure complexes of cutinase with ethyl lactate (EL), the constitutional unit. From this complex structure, the electron density maps clearly showed one lactate (LAC) and one EL occupying different positions in the active site cleft. The binding mode of EL is assumed to show a figure prior to reaction and LAC is an after‐reaction product. These complex structures demonstrate the role of active site residues in the esterase reaction and substrate recognition. The complex structures were compared with other documented complex structures of cutinases and with the structure of PETase from Ideonella sakaiensis. The amino acid residues involved in substrate interaction are highly conserved among these enzymes. Thus, mapping the precise interactions in the Ta_cut and EL complex will pave the way for understanding the plastic‐degrading mechanism of cutinases and suggest ways of creating more potent enzymes by structural protein engineering.

A double mutantEst1, which is a plastic degrading cutinase-type esterase in Thermobifida alba, has been over-expressed in Escherichia coli. The recombinant protein was purified by a two-step protocol involving immobilized metal affinity chromatography and cation-exchange chromatography, yielding 120 mg of protein per liter of bacterial culture. Crystals have been obtained by using the sitting-drop vapor-diffusion technique. Native diffraction data to 1.37 Å resolution were obtained at the BL44XU beam line of SPring-8 from a flash-frozen crystal at 100 K. The crystals belong to space group C2, with unit-cell parameters a = 127.2 Å, b = 42.1 Å, c = 63.2 Å, β = 114.7°, likely containing one Est1 double mutant (296 residues) per asymmetric unit.

The original online version of this article (Kitadokoro, K., Matsui, S., Osokoshi, R., Nakata, K. and Kamitani, S. (2018) Expression, Purification and Crystallization of Thermostable Mutant of Cutinase Est1 from Thermobifida alba. Advances in Bioscience and Biotechnology, 9, 215-223. https://doi.org/10.4236/abb.2018.95015) unfortunately contains some mistakes. The author wishes to correct the errors. Protein Expression and PurificationEst1(A68V/T253P) was used throughout this study (Table 1) [6]. Briefly, 20 mL of an overnight culture of E.coli cells Rosetta-gami B (DE3) transformed with pQE80L-est1(A68V/T253P) was inoculated to 400 mL of LB medium with 50 µg•ml −1 ampicillin. Enzymatic activities were determined by using p-nitrophenylbutyrate (pNPB) ester substrates as previously described [6]. The reaction was performed at 310 K in 1 mL of the mixture containing 50 mM Tris-HCl buffer (pH 8.0), 1 mM pNPB and 0.001 mM enzyme. The reaction mixture without the enzyme was used as the control. Reactions were started by the addition of pNPB.