In this study cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) and Thermobifida fusca DSM44342 (Thf42_Cut1) hydrolyzing poly(ethylene terephthalate) (PET) were successfully cloned and expressed in E.coli BL21-Gold(DE3). Their ability to hydrolyze PET was compared with other enzymes hydrolyzing natural polyesters, including the PHA depolymerase (ePhaZmcl) from Pseudomonas fluorescens and two cutinases from T. fusca KW3. The three isolated Thermobifida cutinases are very similar (only a maximum of 18 amino acid differences) but yet had different kinetic parameters on soluble substrates. Their k cat and K m values on pNP–acetate were in the ranges 2.4–211.9 s–1 and 127–200 μM while on pNP–butyrate they showed k cat and K m values between 5.3 and 195.1 s–1 and between 1483 and 2133 μM. Thc_Cut1 released highest amounts of MHET and terephthalic acid from PET and bis(benzoyloxyethyl) terephthalate (3PET) with the highest concomitant increase in PET hydrophilicity as indicated by water contact angle (WCA) decreases. FTIR-ATR analysis revealed an increase in the crystallinity index A 1340/A 1410 upon enzyme treatment and an increase of the amount of carboxylic and hydroxylic was measured using derivatization with 2-(bromomethyl)naphthalene. Modeling the covalently bound tetrahedral intermediate consisting of cutinase and 3PET indicated that the active site His-209 is in the proximity of the O of the substrate thus allowing hydrolysis. On the other hand, the models indicated that regions of Thc_Cut1 and Thc_Cut2 which differed in electrostatic and in hydrophobic surface properties were able to reach/interact with PET which may explain their different hydrolysis efficiencies.

Hydratases provide access to secondary and tertiary alcohols by regio- and/or stereospecifically adding water to carbon-carbon double bonds. Thereby, hydroxy groups are introduced without the need for costly cofactor recycling, and that makes this approach highly interesting on an industrial scale. Here we present the first crystal structure of a recombinant oleate hydratase originating from Elizabethkingia meningoseptica in the presence of flavin adenine dinucleotide (FAD). A structure-based mutagenesis study targeting active site residues identified E122 and Y241 as crucial for the activation of a water molecule and for protonation of the double bond, respectively. Moreover, we also observed that two-electron reduction of FAD results in a sevenfold increase in the substrate hydration rate. We propose the first reaction mechanism for this enzyme class that explains the requirement for the flavin cofactor and the involvement of conserved amino acid residues in this regio- and stereoselective hydration.

A cutinase from Thermomyces cellullosylitica (Thc_Cut1), hydrolyzing the synthetic polymer polyethylene terephthalate (PET), was fused with two different binding modules to improve sorption and thereby hydrolysis. The binding modules were from cellobiohydrolase I from Hypocrea jecorina (CBM) and from a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (PBM). Although both binding modules have a hydrophobic nature, it was possible to express the proteins in E. coli . Both fusion enzymes and the native one had comparable kcat values in the range of 311 to 342 s(-1) on pNP-butyrate, while the catalytic efficiencies kcat/Km decreased from 0.41 s(-1)/ μM (native enzyme) to 0.21 and 0.33 s(-1)/μM for Thc_Cut1+PBM and Thc_Cut1+CBM, respectively. The fusion enzymes were active both on the insoluble PET model substrate bis(benzoyloxyethyl) terephthalate (3PET) and on PET although the hydrolysis pattern was differed when compared to Thc_Cut1. Enhanced adsorption of the fusion enzymes was visible by chemiluminescence after incubation with a 6xHisTag specific horseradish peroxidase (HRP) labeled probe. Increased adsorption to PET by the fusion enzymes was confirmed with Quarz Crystal Microbalance (QCM-D) analysis and indeed resulted in enhanced hydrolysis activity (3.8× for Thc_Cut1+CBM) on PET, as quantified, based on released mono/oligomers.

The utilization of CO2 as a carbon source for organic synthesis meets the urgent demand for more sustainability in the production of chemicals. Herein, we report on the enzyme‐catalyzed para‐carboxylation of catechols, employing 3,4‐dihydroxybenzoic acid decarboxylases (AroY) that belong to the UbiD enzyme family. Crystal structures and accompanying solution data confirmed that AroY utilizes the recently discovered prenylated FMN (prFMN) cofactor, and requires oxidative maturation to form the catalytically competent prFMNiminium species. This study reports on the in vitro reconstitution and activation of a prFMN‐dependent enzyme that is capable of directly carboxylating aromatic catechol substrates under ambient conditions. A reaction mechanism for the reversible decarboxylation involving an intermediate with a single covalent bond between a quinoid adduct and cofactor is proposed, which is distinct from the mechanism of prFMN‐associated 1,3‐dipolar cycloadditions in related enzymes.