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Polyethylene terephthalate (PET) is a mass‐produced petroleum‐based synthetic polymer. Enzymatic PET degradation using, for example, Ideonella sakaiensis PETase (IsPETase) can be a more environmentally friendly and energy‐saving alternative to the chemical recycling of PET. However, IsPETase is a mesophilic enzyme with an optimal reaction temperature lower than the glass transition temperature (Tg) of PET, where the amorphous polymers can be readily accessed for enzymatic breakdown. In this study, we used error‐prone PCR to generate a mutant library based on a thermostable triple mutant (TM) of IsPETase. The library was screened against the commercially available polyester‐polyurethane Impranil DLN W 50 for more thermostable IsPETase variants, yielding four variants with higher melting points. The most promising IsPETaseTMK95N/F201I variant had a 5.0°C higher melting point than IsPETaseTM. Although this variant showed a slightly lower activity on PET at lower incubation temperatures, its increased thermostability makes it a more active PET hydrolase at higher reaction temperatures up to 60°C. Several other variants were compared and combined with selected previously published IsPETase mutants in terms of thermostability and hydrolytic activity against PET nanoparticles and amorphous PET films. Our findings indicate that thermostability is one of the most important characteristics of an effective PET hydrolase. [ABSTRACT FROM AUTHOR]

Biocatalytic alkylations are important reactions to obtain chemo‐, regio‐ and stereoselectively alkylated compounds. This can be achieved using S‐adenosyl‐l‐methionine (SAM)‐dependent methyltransferases and SAM analogs. It was recently shown that a halide methyltransferase (HMT) from Chloracidobacterium thermophilum can synthesize SAM from SAH and methyl iodide. We developed an iodide‐based assay for the directed evolution of an HMT from Arabidopsis thaliana and used it to identify a V140T variant that can also accept ethyl‐, propyl‐, and allyl iodide to produce the corresponding SAM analogs (90, 50, and 70 % conversion of 15 mg SAH). The V140T AtHMT was used in one‐pot cascades with O‐methyltransferases (IeOMT or COMT) to achieve the regioselective ethylation of luteolin and allylation of 3,4‐dihydroxybenzaldehyde. While a cascade for the propylation of 3,4‐dihydroxybenzaldehyde gave low conversion, the propyl‐SAH intermediate could be confirmed by NMR spectroscopy. [ABSTRACT FROM AUTHOR]

We engineered the cytochrome P450 monooxygenase CYP107D1 (OleP) from Streptomyces antibioticus for the stereo‐ and regioselective 7β‐hydroxylation of lithocholic acid (LCA) to yield ursodeoxycholic acid (UDCA). OleP was previously shown to hydroxylate testosterone at the 7β‐position but LCA is exclusively hydroxylated at the 6β‐position, forming murideoxycholic acid (MDCA). Structural and 3DM analysis, and molecular docking were used to identify amino acid residues F84, S240, and V291 as specificity‐determining residues. Alanine scanning identified S240A as a UDCA‐producing variant. A synthetic "small but smart" library based on these positions was screened using a colorimetric assay for UDCA. We identified a nearly perfectly regio‐ and stereoselective triple mutant (F84Q/S240A/V291G) that produces 10‐fold higher levels of UDCA than the S240A variant. This biocatalyst opens up new possibilities for the environmentally friendly synthesis of UDCA from the biological waste product LCA. [ABSTRACT FROM AUTHOR]