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.

Polyethylene terephthalate (PET) pollution is a global challenge. Advancing the bioprospecting of PET‐degrading enzymes through metagenomics and using computational and functional methods to identify key positions influencing the catalytic rate and selectivity are part of the solution. Here, we report PETase activity in the metagenomic lipase Lip_MRD9, which exhibits peak activity at 30 °C and pH 9.0 and has a denaturation temperature of 42 °C. In addition to acting on long‐chain triglycerides (up to ∼13 units (U)/mg, pH 8.0, 30 °C) and a wide range of 34 other esters (up to ∼228 U/g), Lip_MRD9 hydrolyses mono(2‐hydroxyethyl) terephthalate (∼57 U/g) and bis(2‐hydroxyethyl) terephthalate (∼131 U/g). It also efficiently deconstructs GoodFellow amorphous submicro‐ and nanosized PET particles (∼984/2238 μM degradation products at 30/40 °C, pH 7.0, 21.5 h) and films (∼112/198 μM degradation products at 30/40 °C, pH 7.0, 7 days). Through molecular modelling and experimental analysis, the active site of Lip_MRD9 was revealed, identifying a key residue contributing to its PETase activity compared with those of its closest homologues. This residue plays a crucial role in determining the distinct profiles of degradation products from PET hydrolysis and should be studied in other PETases for its influence on the catalytic process.

We report a method of enzyme stabilisation exploiting the artificial protein chaperone properties of β-cyclodextrin (β-CD) covalently embedded in an ultrathin organosilica layer. Putative interaction points of this artificial chaperone system with the surface of the selected enzyme were studied in silico using a protein energy landscape exploration simulation algorithm. We show that this enzyme shielding method allows for drastic enhancement of enzyme stability under thermal and chemical stress conditions, along with broadening the optimal temperature range of the biocatalyst. The presence of the β-CD macrocycle within the protective layer supports protein refolding after treatment with a surfactant.

Protein engineering is the design and modification of protein structures to optimize their functions or create novel functionalities for applications in biotechnology, medicine or industry. It represents an essential scientific solution for many of the environmental and societal challenges ahead of us, such as polymer degradation. Unlike traditional chemical methods, enzyme-mediated degradation is selective and environmentally friendly and requires milder conditions. Computational methods will play a critical role in developing such solutions by enabling more efficient bioprospecting of natural polymer-degrading enzymes. They provide structural information, generate mechanistic studies, and formulate new hypotheses, facilitating the modeling and modification of these biocatalysts through enzyme engineering. The recent development of pluriZymes constitutes an example, providing a rational mechanism to integrate different biochemical processes into one single enzyme. In this review, we summarize our recent efforts in this line and introduce our early work towards polymer degradation using a pluriZyme-like technology, including our latest development in PET nanoparticle degradation. Moreover, we provide a comprehensive recipe for developing one’s own pluriZyme so that different laboratories can experiment with them and establish new limits. With modest computational resources and with help from this review, your first pluriZyme is one step closer.

The structural complexity of xylan makes its complete degradation challenging. Strategies to improve its hydrolysis often requires enzyme cocktails with multiple specific activities or proteins harboring multiple catalytic domains. Here, we introduce a novel approach through the design of Xyn11_m1, a multifunctional enzyme that combines endoxylanase and feruloyl esterase activities, two catalytic functions involved in the hydrolysis of feruloylated xylans. Using the PluriZyme concept, an artificial feruloyl esterase active site was engineered into the scaffold of a thermophilic glycoside hydrolase family 10 xylanase, Xyn11, from Pseudothermotoga thermarum. Computational design, guided by protein energy landscape exploration simulations, revealed a surface cavity that could accommodate feruloyl-L-arabinose and a xylopentaose (a 5-xylose xylan polymer) bearing a single feruloyl-L-arabinose substitution on the central xylose unit. This cavity was subsequently remodeled into a serine–histidine–aspartic/glutamic acid catalytic triad with feruloyl esterase activity. Molecular dynamics simulations confirmed the stability of the engineered active site. Xyn11_m1 was successfully produced, crystallized, and characterized, and its xylanase activity at 90 °C against oat spelt xylan was comparable to that of the wild-type enzyme (713 ± 4 vs. 600 ± 8 units/mg), and it also displayed feruloyl esterase activity against methyl ferulate (140 ± 5 units/mg), a capability lacking in Xyn11. Notably, Xyn11_m1 exhibited approximately 2.5-fold greater activity compared with Xyn11 (513 ± 27 vs. 222 ± 9 units/mg) against wheat bran xylan containing ferulic acid ester linked to arabinofuranosyl residues. This dual functionality enables efficient degradation of feruloylated xylans, highlighting the potential of PluriZymes to advance biomass deconstruction technologies.

1. Humanity faces the urgent challenge of sustaining social and technological development within planetary boundaries. In response, the United Nations adopted the Sustainable Development Goals (SDG...