The identification or design of biocatalysts to mitigate the accumulation of plastics, including sub-micro- and nano-sized polyethylene terephthalate (nPET), is becoming a global challenge. Here we computationally incorporated two hydrolytic active sites with geometries similar to that of Idionella sakaiensis PET hydrolase, to fragaceatoxin C (FraC), a membrane pore-forming protein. FraC_m1/m2 could be assembled into octameric nanopores (7.0 nm high × 1.6–6.0 nm entry), which deconstructed (40 °C, pH 7.0) nPET from GoodFellow, commodities and plastic bottles. FraC_m1 and FraC_m2 degrade nPET by endo- and exo-type chain scission. While FraC_m1 produces bis(2-hydroxyethyl) terephthalate as the main product, FraC_m2 yields a high diversity of oligomers and terephthalic acid. Mechanistic and biochemical differences with benchmark PET hydrolases, along with pore and nPET dynamics, suggest that these pore-forming protein catalytic nanoreactors do not deconstruct macro-PET but are promising in nanotechnology for filtering, capturing and breaking down nPET, for example, in wastewater treatment plants.

Engineering dual‐function single polypeptide catalysts with two abiotic or biotic catalytic entities (or combinations of both) supporting cascade reactions is becoming an important area of enzyme engineering and catalysis. Herein we present the development of a PluriZyme, TR₂E₂, with efficient native transaminase (k_cat: 69.49±1.77 min⁻¹) and artificial esterase (k_cat: 3908–0.41 min⁻¹) activities integrated into a single scaffold, and evaluate its utility in a cascade reaction. TR₂E₂ (pH_opt: 8.0–9.5; T_opt: 60–65 °C) efficiently converts methyl 3‐oxo‐4‐(2,4,5‐trifluorophenyl)butanoate into 3‐(R)‐amino‐4‐(2,4,5‐trifluorophenyl)butanoic acid, a crucial intermediate for the synthesis of antidiabetic drugs. The reaction proceeds through the conversion of the β‐keto ester into the β‐keto acid at the hydrolytic site and subsequently into the β‐amino acid (e.e. >99 %) at the transaminase site. The catalytic power of the TR₂E₂ PluriZyme was proven with a set of β‐keto esters, demonstrating the potential of such designs to address bioinspired cascade reactions.

Lipases have valuable potential for industrial use, particularly those mostly active against water-insoluble substrates, such as triglycerides composed of long-carbon chain fatty acids. However, in most cases, engineered variants often need to be constructed to achieve optimal performance for such substrates. Protein engineering techniques have been reported as strategies for improving lipase characteristics by introducing specific mutations in the cap domain of esterases or in the lid domain of lipases or through lid domain swapping. Here, we improved the lipase activity of a lipase (WP_075743487.1, or Lip_MRD) retrieved from the Marine Metagenomics MarRef Database and assigned to the Actinoalloteichus genus. The improvement was achieved through site-directed mutagenesis and by substituting its lid domain (FRGTEITQIKDWLTDA) with that of Rhizopus delemar lipase (previously R. oryzae; UniProt accession number, I1BGQ3) (FRGTNSFRSAITDIVF). The results demonstrated that the redesigned mutants gain activity against bulkier triglycerides, such as glyceryl tridecanoate and tridodecanoate, olive oil, coconut oil, and palm oil. Residue W89 (Lip_MRD numbering) appears to be key to the increase in lipase activity, an increase that was also achieved with lid swapping. This study reinforces the importance of the lid domains and their amino acid compositions in determining the substrate specificity of lipases, but the generalization of the lid domain swapping between lipases or the introduction of specific mutations in the lid domain to improve lipase activity may require further investigation.

With advances in protein structure predictions, the number of available high-quality structures has increased dramatically. In light of these advances, structure-based enzyme engineering is expected to become increasingly important for optimizing biocatalysts for industrial processes. Here, we present AsiteDesign, a Monte Carlo-based protocol for structure-based engineering of active sites. AsiteDesign provides a framework for introducing new catalytic residues in a given binding pocket to either create a new catalytic activity or alter the existing one. AsiteDesign is implemented using pyRosetta and incorporates enhanced sampling techniques to efficiently explore the search space. The protocol was tested by designing an alternative catalytic triad in the active site of Pseudomonas fluorescens esterase (PFE). The designed variant was experimentally verified to be active, demonstrating that AsiteDesign can find alternative catalytic triads. Additionally, the AsiteDesign protocol was employed to enhance the hydrolysis of a bulky chiral substrate (1-phenyl-2-pentyl acetate) by PFE. The experimental verification of the designed variants demonstrated that F158L/F198A and F125A/F158L mutations increased the hydrolysis of 1-phenyl-2-pentyl acetate from 8.9 to 66.7 and 23.4%, respectively, and reversed the enantioselectivity of the enzyme from (R) to (S)-enantiopreference, with 32 and 55% enantiomeric excess (ee), respectively.

A peroxygenase‐catalysed hydroxylation of organosilanes is reported. The recombinant peroxygenase from Agrocybe aegerita ( Aae UPO) enabled efficient conversion of a broad range of silane starting materials in attractive productivities (up to 300 mM h ⁻¹ ), catalyst performance (up to 84 s ⁻¹ and more than 120 000 catalytic turnovers). Molecular modelling of the enzyme‐substrate interaction puts a basis for the mechanistic understanding of Aae UPO selectivity.