Fast and sensitive analytical methods are the key to efficient screening of plastic-degrading enzymes. Here, we present a streamlined and affordable approach to assess the enzymatic deconstruction of insoluble synthetic polymers by blending them with a fluorescent dye, rhodamine 6G, and we evaluate this screening method using poly(ethylene terephthalate) (PET) as a model material. Our results indicate that enzymatic depolymerization of the rhodamine-doped PET can be observed in a high-throughput fashion by following release of the fluorophore. The fluorescence data obtained during the hydrolysis of rhodamine-doped PET by 14 PET hydrolases, produced with a robotic platform, correlated with the quantitative chromatographic analysis of PET degradation products. Remarkably, the use of the rhodamine-loaded PET substrate resulted in negligibly low background signals even when detecting PETase activity in crude cell lysates, suggesting suitability for screening of a wide variety of samples. Encouraged by these results, we next produced a selection of polyethylene- and nylon-based materials loaded with rhodamine 6G. While rapid leaching of fluorophore observed with nylon substrates limits the utility of the method for detecting nylonase activity, the rhodamine-loaded polyethylene showed promising performance in passive diffusion tests, indicating that this latter substrate may be used to screen for polyolefin-degrading enzymes.

Abstract The large-scale production of citric acid using Aspergillus niger generates substantial quantities of fungal biomass, which remains an underutilized resource. In this study, we developed a tailored enzyme-based strategy to valorize A. niger biomass by selectively extracting chitin, while generating glucose-rich hydrolyzates suitable for fermentation. Comprehensive screening of 16 commercial proteases and glucanases led to the identification of optimal enzyme combinations and process conditions. At a 30 L scale, this process yielded a chitin-rich fraction representing 27% of the initial biomass. The extracted chitin was characterized using X-ray diffraction and 13C solid-state nuclear magnetic resonance (NMR) spectroscopy, revealing high crystallinity and purity. The chitin content of the chitin-rich fraction was quantified using newly established NMR calibration curves based on distinct carbon signals associated with the acetamido group (C-2, CH3, and C═O) and estimated to be on the order of 74%. The chitin-rich fraction was subsequently converted into chitosan with a degree of deacetylation of ∼55% (45% yield on a dry matter basis) and colloidal chitin (60% yield on a dry matter basis), with product properties similar to those of corresponding products derived from crustacean chitin. This study demonstrates the feasibility of enzyme-only extraction of fungal chitin and provides a scalable, sustainable approach for converting fungal biomass into high-value bioproducts.

Chitin is a ubiquitous structural biopolymer with significant potential for various high-value applications. Chitin deacetylases (CDAs) can potentially deacetylate chitinous molecules, producing chitosans and partially acetylated chitooligosaccharides (COS). Here, we describe the expression and characterization of a CDA from the filamentous fungus Absidia coerulea, AcCDA1, active toward colloidal chitin, partially deacetylated chitin, chitosan with different degrees of acetylation, and COS. When incubated with COS with degrees of polymerization 3-6, the enzyme rapidly produced fully deacetylated COS, which is a relatively rare property among known CDAs. The mode of action of AcCDA1 toward penta-N-acetyl chitopentaose ((GlcNAc)5) was studied by mass spectrometry, using a novel approach for labeling the reducing N-acetyl-D-glucosamine unit of the COS with chitooligosaccharide oxidase (FgChitO) from Fusarium graminearum. Kinetic studies with COS revealed high enzyme efficiency with kcat/Km values reaching 18.8 mM-1 s-1 for (GlcNAc)5. The enzyme removed 12% of the acetyl groups in colloidal chitin whereas it drastically reduced the degree of acetylation of various chitosans. A comparative study with three other CDAs showed that AcCDA1 is highly efficient enzyme and revealed both similarities and differences. These findings show that various CDAs can achieve different deacetylation targets during the enzymatic processing of chitinous materials.

Efficient enzymatic depolymerization of recalcitrant polysaccharides such as chitin and cellulose relies on processive glycoside hydrolases (GHs), whose efficiency can be enhanced through cooperation with lytic polysaccharide monooxygenases (LPMOs). For processive GHs, retained binding during successive catalytic events aids depolymerization of crystalline substrates but comes at the expense of slow dissociation (low k off) that limits turnover. Here, we engineered a series of mutants of the processive exochitobiohydrolase SmChiB from Serratia marcescens, in which a major determinant of substrate affinity and processivity, Trp220, was replaced by Tyr, Phe, His, Gln, or Ala. Functional analysis showed that these mutants had stepwise reductions in substrate affinity and processivity, with the latter being a signature of an increased k off. Molecular dynamics simulations confirmed that Trp220 plays an important role in substrate binding. The less processive SmChiB variants, and in particular W220Y, were able to reach wild-type-like performance only at high substrate concentrations. Importantly, the use of LPMOs to decrystallize the substrate and thereby increase its effective concentration, enhanced the performance of the less processive mutants to a much greater extent than for wild-type SmChiB. In some reaction setups, the combination of W220Y with an LPMO yielded twice as much soluble product compared to the wild-type enzyme under identical conditions. Thus, when combined with LPMOs, less processive GHs become more favorable because they are intrinsically more efficient catalysts when acting on noncrystalline substrates. These findings shed light on how the interplay between GHs and LPMOs can be optimized for efficient enzymatic conversion of recalcitrant polysaccharides.

Bacterial two-domain multicopper oxidases (2dMCOs) represent a structurally distinct class of trimeric multicopper oxidases. Differing considerably from well-characterized monomeric three-domain laccases, type B 2dMCOs have a T1 active site positioned in a tunnel in the trimer center. The biochemical properties and roles in lignin conversion of 2dMCOs remain poorly understood. Here, we present a comprehensive biochemical characterization of a type B 2dMCO from Cellvibrio japonicus (CjMCO) and discuss links between its structural organization and activity. The T1 copper of CjMCO had a redox potential of 537 mV, but the turnover number (0.4 s-1) was ∼1000-fold lower than high-turnover fungal laccases. Stopped-flow UV-vis spectroscopy indicated that this low turnover likely reflects slow reoxidation of the enzyme by O2, which has not been previously reported for laccases. Despite the steric constraints imposed by its trimeric structure, CjMCO oxidized the lignin model compound guaiacylglycerol-β-guaiacyl ether, resulting in both oxidative coupling and bond cleavage, and CjMCO was able to act on oligomeric birch organosolv lignin, promoting net oxidative polymerization. Interestingly, 2,6-dimethoxyphenol oxidation kinetics and the product profile for guaiacylglycerol-β-guaiacyl ether oxidation by CjMCO were influenced by pH, buffer composition, and ionic strength, suggesting a potential strategy for tailoring product profiles. Together, these findings demonstrated that CjMCO functions as a laccase and oxidizes phenolic lignin moieties, but its slow rates and trimeric architecture indicate that it is unlikely to efficiently degrade lignin polymers in vivo. This study expands the current understanding of bacterial laccase diversity and provides a foundation for exploring other physiological roles of type B 2dMCOs beyond lignin degradation.

In this issue of Chem Catalysis, Westh and co-workers use linear free-energy relationships and an impressive and unique amount of enzyme kinetic data to understand rate-limiting steps in cellulose hydrolysis by cellulases. The authors provide a conceptual framework for describing, understanding, and optimizing interfacial enzyme catalysis.

Enzymes known as lytic polysaccharide monooxygenases (LPMOs) are exceptionally powerful small redox enzymes that master the controlled generation and productive use of potentially damaging hydroxyl radicals in what is essentially a H2O2-driven peroxygenase reaction. We have used ancestral sequence reconstruction and enzyme resurrection to unravel evolutionary steps leading to this exceptional catalytic ability. Real-time monitoring of copper reoxidation and amino acid radical formation showed evolutionary improvement of both the capacity to avoid futile turnover of H2O2 and the ability to scavenge damaging radicals resulting from such turnover through a hole hopping pathway. Through mutational studies of ancestral LPMOs, we show that adoption of an extant-like conformation of residues in the hole hopping pathway yields improvements in redox robustness to near-extant levels. These results show how selective pressure imposed by the need for generating a highly oxidizing intermediate is a key driver of metalloenzyme evolution, involving large parts of the enzyme, well beyond the catalytic center.

Expansins and expansin-like proteins, which together comprise the expansin superfamily, are small cell wall (CW)-loosening proteins present in diverse organisms, including plants, fungi, bacteria, protists, molluscs and nematodes. Proteins belonging to the expansin superfamily (ESPs) have diverse biological roles, including enabling CW growth, serving as virulence factor for plant pathogens, and assisting plant CW decomposition by microbial plant CW-active enzymes. In this review, we offer a biomechanical perspective of the role of ESPs, by providing an overview of the CW properties they affect, examining their reported substrate-binding affinities and assessing how these relate to the functions of the respective CW components. Moreover, we refine the current nomenclature of ESPs. Finally, within the framework of our recent discovery of catalytic activity in an expansin-like protein, we itemise biomechanical and biochemical assays that are commonly employed to evaluate activity of ESPs, and we discuss challenges associated with determining ESP activity. We hope that this review will promote research and open new research avenues in the field of ESPs.

Lytic polysaccharide monooxygenases (LPMOs) catalyze oxidative cleavage of glycosidic bonds and are important members of Nature's enzymatic machinery for the deconstruction of recalcitrant polysaccharides. While chitin-active LPMOs are typically found in microorganisms that also produce chitinases, Lactiplantibacillus plantarum, a commensal lactic acid bacterium, lacks the chitinolytic machinery yet encodes a single-domain LPMO, hereinafter called LpLPMO10A. Here, we characterize LpLPMO10A produced both homologously and heterologously and demonstrate through structural modeling and sequence comparisons that it groups with canonical chitin-active LPMOs. Biochemical assays show that LpLPMO10A catalyzes oxidative cleavage of chitin, producing C1-oxidized products. Analysis of productive (chitin peroxygenase reaction) and nonproductive (ascorbate peroxidase reaction) H2O2-consumption, together with additional functional assays, showed that LpLPMO10A is very similar to SmLPMO10A from Serratia marcescens, with a known role in chitin degradation. Moreover, LpLPMO10A was more efficient in degrading chitin than BlLPMO10A from Bacillus licheniformis, which harbors a chitinolytic gene repertoire including chitinases. For all three LPMOs, these activities showed only minor pH-dependence in the pH 5 - 7 range, meaning that LpLPMO10A does not seem to have a particular potential to act at acidic pH. This lack of pH dependence is shared with fungal LPMOs despite the presence of different titratable groups in the second coordination sphere of the copper. While our results reveal functional variation among chitin-active bacterial LPMOs, for example regarding their ability to withstand oxidative damage, they do not identify distinct functional features that clarify the biological role of LpLPMO10A. Taken together, our findings highlight LpLPMO10A as an evolutionarily intriguing LPMO, namely a chitin-active enzyme retained in a nonchitinolytic bacterium.

Expansin-related proteins (ERPs) are a broad group of plant cell wall-loosening proteins and are considered noncatalytic, as, to date, no cell wall-derived products have been observed as a result of catalysis, despite the presence of a domain that resembles the catalytic domains of GH45 endoglucanases. Here, we report catalytic activity for a single-domain ERP, GtEXPN_133317, from the brown-rot fungus Gloeophyllum trabeum, which is highly expressed in the early phase of spruce colonization. We demonstrate enzyme-dependent formation of xylan-derived products, such as glucuronylated xylo-oligosaccharides, using high-performance anion exchange chromatography with pulsed amperometric detection. Structure-based multiple sequence alignment of ERPs with GH45 endoglucanases showed that, next to a single conserved aspartate (Asp87 in GtEXPN_133317) present in all ERPs and GH45s, fungal ERPs contain a second conserved acidic residue (Asp25 in GtEXPN_133317). Mutation of these two conserved amino acids, Asp87 and Asp25, led to a nearly complete loss of xylanolytic activity. While these findings do not exclude the possibility of a noncatalytic plant cell wall-loosening mechanism, they show that ERPs likely have other modes of action besides what the current paradigm states.

With this status report, we aim to provide a timely snapshot of the protein engineering field as a broad and rapidly advancing discipline that integrates computational, molecular biology, structure-guided, evolutionary, and synthetic approaches to create new and improved proteins with tailored structures and useful functions. The report is organized into eight thematic areas spanning core methodologies and major application domains, including enzymes, therapeutics, detection, synthetic biology, and materials. Contributions from experts across these areas highlight both the historical foundations and recent advances in their respective fields, with particular emphasis on the growing influence of machine learning and artificial intelligence-based methods. Emerging from this broad overview is a central message: protein engineering appears to be entering a golden age, defined by a rapidly accelerating pace of progress, even as significant challenges in design, screening, and real-world application remain. Looking ahead, the continued integration of computational and experimental strategies is poised to further accelerate the impact of protein engineering across an expanding range of economically and societally important sectors, from therapeutics and molecular imaging to diagnostics, plastic recycling, and industrial chemistry.

Lignin is the most abundant renewable source of aromatic carbon and its microbial depolymerization and metabolism under aerobic conditions is well studied. However, lignin breakdown in the absence of oxygen remains poorly understood. In this study, we established long-term bacterial enrichment cultures supplied with diverse lignin preparations as the sole carbon source under denitrifying conditions. Denitrification dynamics were followed by monitoring nitrogenous gases. Metagenomics analysis of eight enrichments involving five lignins recovered 62 metagenome-assembled genomes (MAGs), several of which encoded enzymes for both denitrification and anaerobic metabolism of aromatic compounds. Quantitative metaproteomics confirmed expression of such enzymes and additionally showed that several MAGs expressed multiple oxidoreductases and uncharacterised proteins that are potential candidates for involvement in lignin modification. The detection of several oxygen-dependent oxidoreductases despite anaerobic conditions prompts intriguing discussion of potential mechanistic explanations. This systems-level study expands our understanding of bacterial processing of lignin-associated carbon in anaerobic environments and suggests enzymatic targets for further exploration of lignin depolymerization under oxygen-limited conditions.

Unspecific peroxygenases (UPOs, EC 1.11.2.1) are promising biocatalysts for the oxyfunctionalization of organic molecules and the synthesis of industrially relevant compounds because of their vast repertoire of catalyzed reactions. To date, thousands of putative UPO genes have been identified in eukaryotic genomes, most of them in the Ascomycota and Basidiomycota phyla, and several UPOs have been characterized. Remarkably, no related enzymes have been reported in prokaryotic organisms. Here, we describe the discovery of a diverse family of bacterial heme-thiolate peroxygenases through structure database mining, followed by functional characterization of selected representatives. The bacterial proteins, termed bacterial UPOs (BUPOs), display clear structural homology to family I (short) fungal UPOs despite low sequence identity (<20%). Expression of one of these proteins (HydBUPO) in its native host (Hydrogenophagasp. A37) was confirmed by proteomics. Several BUPOs were cloned and expressed in Escherichia coli. In biochemical assays, the BUPOs were able to catalyze one-electron oxidation (peroxidase activity) of ABTS and 2,6-dimethoxyphenol, as well as two-electron oxidation (peroxygenase activity) of naphthalene, indole, 3-phenyl-1-propanol, and 16-hydroxypalmitic acid, using hydrogen peroxide as a cosubstrate. These enzymes thus represent a previously unknown group of bacterial heme-thiolate peroxygenases that share key structural and functional features with eukaryotic UPOs, offering potential candidates for the development of industrially relevant biocatalysts.

Onygena corvina is a non-pathogenic, saprophytic fungus that colonizes feathers, hooves, and hair, and represents a valuable source of keratin-degrading enzymes. The only genome assembly of O. corvina available to date was obtained for the strain CBS 281.48 using Illumina short-read sequencing, yielding a reference genome composed of 521 contigs with a contig N50 of 0.229 Mb. Here, we report an improved O. corvina CBS 281.48 genome assembly generated using a high-quality hybrid approach that combines Illumina short-read and Oxford Nanopore long-read sequencing. The new assembly consists of only 13 contigs totaling 21.8 Mb, with an N50 of 4.4 Mb, and has a completeness of 98.4

BACKGROUND:Feathers are a major by-product of the poultry industry, which poses an environmental challenge due to the recalcitrant structure of keratin, making them resistant to degradation. Traditional methods of feather handling, like conversion to feather meal, are energy-intensive and have limited efficiency. Biotechnological approaches, particularly microbial hydrolysis, offer a novel and more sustainable alternative for keratin degradation. This study evaluated feather hydrolysis by two bacterial strains, newly characterized cold-adapted Arthrobacter oryzae (BIM B-1663) and Bacillus licheniformis (CCM 2145T), known as a keratin degrader, under various feather pre-treatment conditions, including washing, autoclaving, drying, and grinding. RESULTS:Both bacterial strains were able to degrade pretreated feathers with a degradation efficiency of 75 to 90%, resulting in high ratios of nitrogen to carbon in the hydrolysates. B. licheniformis confirmed its enzymatic capabilities with high levels of general and specific protease activity and furthermore presented enriched amounts of amino acids of industrial interest. A. oryzae showed a much higher keratinase/protease activity ratio, demonstrating high specificity and efficiency of its enzymes. Autoclaving emerged as the most important determinant of microbial degradation efficiency and influenced the composition (peptide pattern, amino acid content, and chemical composition assessed through FTIR) of the resulting hydrolysates. Feather drying, although not improving microbial degradation efficiencies, had a considerable impact on hydrolysate composition. CONCLUSIONS:The results show that both tested bacterial strains can efficiently degrade autoclaved feathers but use distinct enzymatic strategies to do so. Enriched profiles in amino acids and high nitrogen content in the hydrolysates also advocate for the benefits of microbial feather hydrolysis over an enzymatic one. To the authors' knowledge this study is the first to report a comprehensive evaluation of the impact of various feather pre-treatment methods on the efficiency of subsequent microbial feather hydrolysis and is the first one to report enrichment in phenylalanine, lysine, and tyrosine secreted by B. licheniformis.

Lactic acid bacteria, such as Lactiplantibacillus plantarum, are becoming increasingly popular hosts for combining production and delivery of therapeutic proteins to immune cells. Soluble antigens are susceptible to rapid proteolysis, hence anchoring of antigens to bacterial cells, which likely protects the antigen, is a preferred delivery strategy that may increase immune responses. In cancer research, personalized immunotherapy has high potential and, in this respect, the so-called neoantigens that accumulate in tumor cells are promising tumor specific targets. Here, we demonstrate that, when using the inducible pSIP expression system, L. plantarum can produce and surface display two neoantigens, NAG1 and ETV6. The antigens could be targeted to both the cell membrane and the cell wall, utilizing four different anchoring methods. The production level and the degree of surface exposure of the antigens varied among the anchors. Flow cytometry analysis showed that antigens anchored to the cell wall were more exposed than those anchored to the cell membrane. To our knowledge these are the first reports of neoantigens being produced and surface-displayed in Lactobacillales.

Lactiplantibacillus plantarum and other lactic acid bacteria are emerging as promising candidates for mucosal delivery of surface-displayed antigens. However, producing secreted heterologous proteins and anchoring these using LPxTG anchors can significantly reduce bacterial fitness. To understand the underlying mechanisms and limiting factors, we analyzed 11 recombinant L. plantarum strains expressing the HaloTag reporter protein with the same LPxTG anchor but varying signal peptides. By labeling the reporter protein with fluorescent ligands, this approach allowed simultaneous detection of correctly folded intracellular and extracellular protein, revealing signal peptide-dependent variation in the relative amounts of intra- and extracellularly folded HaloTag. Furthermore, this analysis uncovered an unexpected correlation between secretion efficiency and bacterial fitness. Strains with better growth showed more premature intracellular folding and reduced protein translocation and surface display. Conversely, strains with a higher fraction of surface-displayed protein, i.e. strains with greater secretion efficiency, exhibited impaired growth, likely due to increased interactions between the signal peptide and the secretion machinery, leading to secretion overload. Correlation analyses and confirmation of observed correlations by mutational studies of the signal peptides showed that signal peptide hydrophobicity is positively correlated with higher secretion efficiency but is accompanied by a trade-off in fitness. These findings underscore the critical role of signal peptides in balancing protein secretion and bacterial viability, offering valuable insights for optimizing protein secretion and anchoring in gram-positive bacteria.

Unspecific peroxygenases (UPOs) are versatile enzymes capable of oxidizing a broad range of substrates, using hydrogen peroxide as the sole co-substrate. In this study, UPOs were evaluated for their potential in the selective oxyfunctionalization of the phenolic lignin monomer 4-propylguaiacol (4-PG) to generate versatile scaffolds for the synthesis of high-value compounds. In addition to the desired peroxygenase reaction, the phenolic group of 4-PG is susceptible to undesirable one-electron oxidation (peroxidase activity). Assessment of the activity of 19 UPOs from phylogenetically diverse clades toward 4-PG revealed that several UPOs could serve as potential biocatalysts for the functionalization of 4-PG, with some enzymes showing both promising conversion yields (>50%) and regioselectivity for the peroxygenase reaction. Pronounced differences in peroxygenase:peroxidase activity ratios and regioselectivity were observed. Comparative analysis-supported by experimental activity profiles and structural data-suggest that a more constrained active-site topology contributes to the peroxygenase activity. UPOs from a clade within the Ascomycota phylum with high peroxygenase activity possess a unique aliphatic pocket in their catalytic centers. Our study provides valuable insights into the structure-function relationships underpinning enhanced peroxygenase activity of UPOs and provides a functional mapping of a broad UPO-sequence space for 4-PG, highlighting these enzymes as promising catalysts for the selective oxyfunctionalization of a phenolic lignin monomer.

Several bacterial pathogens secrete multidomain enzymes known as lytic polysaccharide monooxygenases (LPMOs) that are important for virulence. One example is the Vibrio cholerae virulence factor GbpA (VcGbpA), in which an N-terminal LPMO domain is followed by two domains of unknown function called GbpA2 and GbpA3, and a C-terminal chitin-binding domain called CBM73. In-depth functional characterization of full-length and truncated variants of VcGbpA and a homologue from V. campbellii (previously V. harveyi, VhGbpA) showed that the catalytic LPMO domains of these proteins exhibit properties similar to natural single-domain LPMOs with established roles in chitin degradation. Interestingly, binding to chitin and efficient degradation of this substrate were affected by the presence of the GbpA2 and GbpA3 domains. Combined with structural predictions and analyses of sequence conservation, our data show that GbpA3 has evolved to interact with the reduced catalytic copper site in the LPMO domain to prevent off-pathway reactions in the absence of substrate. Substrate binding by CBM73 weakens this interaction, enabling the activation of the LPMO only when substrate is present. These observations shed new light into the functionality of these multidomain LPMOs and uncover a novel mechanism for regulating LPMO activity.