Poly(butylene adipate-co-terephthalate) (PBAT), a polyester made of terephthalic acid (TPA), 1,4-butanediol, and adipic acid, is extensively utilized in plastic production and has accumulated globally as environmental waste. Biodegradation is an attractive strategy to manage PBAT, but an effective PBAT-degrading enzyme is required. Here, we demonstrate that cutinases are highly potent enzymes that can completely decompose PBAT films in 48 h. We further show that the engineered cutinases, by applying a double mutation strategy to render a more flexible substrate-binding pocket exhibit higher decomposition rates. Notably, these variants produce TPA as a major end-product, which is beneficial feature for the future recycling economy. The crystal structures of wild type and double mutation of a cutinase from Thermobifida fusca in complex with a substrate analogue are also solved, elucidating their substrate-binding modes. These structural and biochemical analyses enable us to propose the mechanism of cutinase-mediated PBAT degradation.

The biosynthesis of neurotoxin aetokthonotoxin (AETX) that features a unique structure of pentabrominated biindole nitrile involves a first-of-its-kind nitrile synthase termed AetD, an enzyme that shares very low sequence identity to known structures and catalyzes an unprecedented mechanism. In this study, we resolve the crystal structure of AetD in complex with the substrate 5,7-di-Br-L-Trp. AetD adopts the heme oxygenase like fold and forms a hydrophobic cavity within a helical bundle to accommodate the indole moiety. A diiron cluster comprising two irons that serves as a catalytic center binds to the carboxyl O and the amino N of the substrate. Notably, we demonstrate that the AetD-catalyzed reaction is independent of the bromination of the substrate and also solved crystal structures of AetD in complex with 5-Br-L-Trp and L-Trp. Altogether, the present study reveals the substrate-binding pattern and validates the diiron cluster-comprising active center of AetD, which should provide important basis to support the mechanistic investigations into this class of nitrile synthase.

Calycosin-7-O-β-D-glucoside (Cy7G) is one of the principal components of Radix astragali. This isoflavonoid glucoside is regarded as an indicator to assess the quality of R. astragali and exhibits diverse pharmacological activities. In this study, uridine diphosphate-dependent glucosyltransferase (UGT) UGT88E18 was isolated from Glycine max and expressed in Escherichia coli. Recombinant UGT88E18 could selectively and effectively glucosylate the C7 hydroxyl group of calycosin to synthesize Cy7G. A one-pot reaction by coupling UGT88E18 to sucrose synthase (SuSy) from G. max was developed. The UGT88E18–SuSy cascade reaction could recycle the costly uridine diphosphate glucose (UDPG) from cheap sucrose and catalytic amounts of uridine diphosphate (UDP). The important factors for UGT88E18–SuSy cascade reaction, including UGT88E18/SuSy ratios, different temperatures, and pH values, different concentrations of dimethyl sulfoxide (DMSO), UDP, sucrose, and calycosin, were optimized. We produced 10.5 g L⁻¹ Cy7G in the optimal reaction conditions by the stepwise addition of calycosin. The molar conversion of calycosin was 97.5%, with a space–time yield of 747 mg L⁻¹ h⁻¹ and a UDPG recycle of 78 times. The present study provides a new avenue for the efficient and cost-effective semisynthesis of Cy7G and other valuable isoflavonoid glucosides by UGT–SuSy cascade reaction.