The severe, long-lasting harm caused by plastic pollution to marine ecosystems and coastal economies has led to the development of biodegradable plastics; however, their limited decomposition in marine environments remains a challenge. Here, technologies are presented for creating 3D-bioprinted living materials as a proof of concept for bioplastic degradation, with specific use in marine environments. The approach developed here integrates the halotolerant bioplastic-degrading bacterium Bacillus sp. NRRL B-14911 into alginate-based bio-ink to print an engineered living material (ELM) termed a "bio-sticker." Quantification of bacteria viability reveals that bioprinted marine bacteria survive within biostickers for more than 3 weeks. The rate at which the biostickers degrade the bioplastic polyhydroxybutyrate (PHB) can be tuned by altering biosticker biomass concentration, bioplastic concentration, or incubation temperature. Biostickers that are transferred to a different PHB sample still retain high biodegradation activity, demonstrating their reusability. Strain sweep oscillatory tests demonstrate that the biostickers display predominantly viscoelastic behavior. Monotonic tensile tests indicate that the elastic modulus and the adhesion of the biostickers are not negatively impacted by bacteria growth or incubation temperature. This work paves the way for the development of ELMs to facilitate the inclusion of bioplastics within the blue economy, promoting the emergence of more sustainable and eco-friendly materials.

Recycling of enzymatically depolymerized poly(ethylene terephthalate) (PET) involves polycondensation of bis(2-hydroxy-ethyl) terephthalic acid (BHET)-a degradation product of enzymatic PET hydrolysis. The recycling process is simplified when more BHET is generated by the enzymatic reaction. Here, we report how ethylene glycol (EG) addition can maximize BHET formation using leading PET hydrolases, LCCICCG, and PHL7. EG at any level above 2-5% vol/vol was found to decrease the steady-state enzymatic degradation rates while enhancing the relative production of BHET. For LCCICCG, the highest measured BHET levels (product fraction approaching 0.5) were attained at EG levels of ∼27-29% and reaction temperature ∼62.5°C. EG shortened the enzymatic reaction lag-phase and lowered the lag-phase increase with PET crystallinity. EG works by perturbing the adsorption, including nonproductive adsorption, of the enzymes to the PET surface, which manifests as an apparent change in substrate affinity (increases the invKm in interfacial kinetics modeling) and directs the enzyme more to the liquid phase.

We present the development and comprehensive characterization of a biocatalytic system comprising laccase immobilized on a polystyrene-chitosan (PS-chitosan) carrier. The material was synthesized using electrospinning, which enabled the formation of a porous structure with a large surface area, offering favorable properties. Laccase from Trametes versicolor was successfully immobilized on the PS-chitosan with a yield of 87% and an activity retention of 83%. The immobilization was assessed by means of Fourier transform infrared spectroscopy (FTIR), confocal laser scanning microscopy (CLSM), electrokinetic potential measurements, and scanning electron microscopy (SEM). Kinetic analyses and thermodynamic studies confirmed that immobilization favorably influenced the catalytic properties of the laccase. Molecular docking and molecular dynamics simulations enabled identification of the preferred sites for binding of the enzyme to the material, as well as visualization of the interaction between the immobilized enzyme and the estrogen 17α-ethinylestradiol (EE2) substrate. The system enabled the removal of EE2 from model samples and real wastewater, achieving removal efficiencies of 86% and 44%, respectively. Overall, the study provides new insight into the immobilization binding of laccase to a PS-chitosan support and confirms the bioremediation potential of laccase immobilized on PS-chitosan, paving the way for developing efficient biocatalytic systems.

Feruloylated xylooligosaccharides (FXOS) were alkali-solubilized from corn bran followed by enzymatic treatments (4 enzyme treatments were compared). The FXOS were ultrafiltrated using molecular weight cut-offs of 1 kDa and 10 kDa. Antioxidant activities, α-amylase and α-glucosidase inhibitory effects were assessed for the different fractions. The lower molecular weight fraction (<1 kDa), enzymatically hydrolyzed for 24 h (FXOS1-24), exhibited the strongest in vitro antioxidant activity, with IC50 values of 0.14 mg/mL (DPPH) and 1.0 mg/mL (ABTS•+). In addition, FXOS1-24 inhibited porcine pancreatic α-amylase (IC50 ∼ 13 mg/mL) and showed significant α-glucosidase inhibitory activity, suggesting potential antidiabetic properties. The bioactivities correlated positively to ferulic acid content of the solubilized xylooligosaccharides. The findings demonstrate new functional effects of soluble feruloylated xylooligosaccharides that yield new opportunities for upgrading of corn bran from corn starch processing.

Ulvan is a polysaccharide most abundant in green macroalgae biomass. Investigation of ulvan confirmed the potential of the polysaccharide for food, pharmaceutical and chemistry applications, emphasising the beneficial properties of ulvan oligosaccharides. Efficient production of oligosaccharides requires action of ulvan lyases capable of ensuring polysaccharide enzymatic depolymerisation. The armoury of available ulvan lyases was expanded by characterisation of SH2L_Ulv3 ulvan lyase, which was found to be phylogenetically distinct from previously characterised lyases attributed to PL25 family. A gene encoding a novel ulvan lyase was identified among sequences from a seaweed biomass metagenome enriched in an intertidal coastal hot spring. Identified ulvan lyase was most similar to a hypothetical protein from a Bacteroidales bacterium. Recombinant SH2L_Ulv3 was heterologously (over)produced in Escherichia coli at a high yield, remaining soluble in the expression host as well as after affinity purification. Ulvan lyase active as a 48.6 kDa monomer with evaluated activity optimum pH 7.5 and 200 mm NaCl at 25 °C demonstrated broad substrate specificity. SH2L_Ulv3 degraded ulvan from blade-thallus as well as tubular-thallus morphology algae species, efficiently producing three different DP4 and DP2 unsaturated oligosaccharides. The kinetic parameters of SH2L_Ulv3 were KM 3.63 ± 0.12 mg·mL-1, Vmax 1.78 ± 0.04 μmol·min-1·mL-1 and kcat 1.46 ± 0.04 s-1. Magnesium ion stimulated SH2L_Ulv3 activity. The characterised enzyme was not thermostable, displaying Tm 42 °C. The computationally modelled structure of SH2L_Ulv3 revealed structural organisation and active site architecture as well as ligand substrate binding and zinc ion coordinating residues typical for PL25 lyases; however, with a larger central active site cleft facilitating ulvan polysaccharide degradation.

Dps is the most abundant nucleoid-associated protein in starved Escherichia coli with ∼180 000 copies per cell. Dps binds DNA and oxidizes iron, facilitating survival in harsh environments. Dps-DNA complexes can form crystalline structures, leading to the proposed model that Dps reorganizes the starved E. coli nucleoid into a compact liquid crystal, slowing chromosome dynamics, and limiting access of other proteins to DNA. In this work, we directly tested this model using live-cell super-resolution microscopy and Hi-C analysis. We found that after 96 h of starvation, Dps compacts the nucleoid, and increases short-range DNA-DNA interactions but does not affect chromosome accessibility to large protein nanocages or small restriction enzymes. We also report that chromosome dynamics and organization are primarily impacted by the bacterial growth phase; the effect of Dps is relatively minor. Our work clarifies the role of Dps in modulating nucleoid properties, and we propose an updated model for Dps-DNA interactions in which Dps binds, protects, and compacts DNA largely without influencing chromosome access, dynamics, and organization. Additionally, this work provides a general framework for assessing the impact of nucleoid-associated proteins on key aspects of chromosome function in live cells.

Disulfide reductases can cleave disulfide bonds that interlock with the keratin structures to promote keratin biomass degradation by keratinolytic proteases. Yet, the synergy between purified keratinolytic proteases and disulfide reductases remains underexplored. The study identified a keratinolytic protease, BrgM4, and a thioredoxin reductase, BrgTrxR, from the keratinolytic Brevibacillus gelatini LD5. The BrgM4 exhibited high keratinolytic activity at 90 °C and pH 7.5 and had remarkable thermostability (t1/2 = 13.6 h at 65 °C). The BrgM4 and BrgTrxR showed synergism in degrading chicken feathers at 50 °C, pH 6.5. BrgM4 preferentially cleaves feather keratin at the C-terminus of Thr, Val, Pro, Arg, and Ala. Beyond the active site residues, the amino acids Asn106, Ala107, Trp109, Arg192, Asp215, His220, and Zn2+ are important for BrgM4-substrate binding. These findings provide insights into the BrgM4 function and synergistic catalytic activity of keratinolytic protease and reductase, guiding the design of enzyme cocktails for efficient keratin conversion.

3D printing has revolutionized the field of tissue engineering and regenerative medicine, emerging as a widely adoptable strategy for the fabrication of mammalian cell-laden constructs laden with complex microenvironments. More recently, 3D printed living materials containing microorganisms have been developed. The potential for engineered 3D living materials as in vitro models for biomedical applications, such as antimicrobial susceptibility testing, is extensive; however, the need for an in-depth understanding of the relationship between the complex construct and the microorganism response still exists. Additionally, there exists a lack of multispecies engineered living material models (ELMM), which more closely mimic naturally occurring biofilms. This work includes the successful development of 3D printed single and mixed species in vitro ELMM for the development of antimicrobial therapeutics. Results successfully demonstrated the effect of maturation age on response to antimicrobial agents. Additionally, a gelatin 3D printing bath was fabricated, characterized, and yielded biomimetic 3D ELMM that could not otherwise be fabricated with low viscosity bioinks. With (1) non-traditional scaffold fabrication techniques for low viscosity bioinks, (2) enhanced understanding of the effect of biofilm maturation age on antimicrobial susceptibility, and (3) investigation into the interaction of mixed species models, 3D printed engineered living materials could provide in vitro infectious disease models for the discovery of distinct antibiofilm drugs. The results show proof-of-concept in vitro multispecies ELMM to more accurately mimic naturally occurring conditions with confirmed cell viability and maturation.

Lignin is the largest renewable resource for aromatics, and the quest to understand enzymatic lignin modification has never been more important. A recently recognized group of single-domain type-3 copper enzymes, named ortho-methoxyphenolases (o-MPs, EC 1.14.18.13) and previously referred to as short polyphenol oxidases (PPOs), found in filamentous fungi can sequentially o-hydroxylate and oxidize guaiacyl-type phenols into methoxy-o-quinones. A subset of these enzymes also targets syringyl-type phenols and, via an unprecedented oxidative o-demethoxylation mechanism, funnels these into the same methoxy-o-quinones generated from guaiacyl-type compounds. Here, we demonstrate that fungal o-methoxyphenolases also cleave bonds in lignin model dimers representing the abundant β-O-4'-linked substructures of lignin, having guaiacyl and, in some cases, syringyl terminal phenolic groups. Based on advanced liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR) analysis, and isotope labeling, we propose a mechanism in which the enzymatic formation of methoxy-o-quinone moieties in the model dimers triggers intramolecular rearrangements that lead to different types of bond cleavage, where C1-Cα cleavage predominates. Additionally, β-ether breakage and formation of Cα-ketone groups occur. We investigate the influence of pH and reductants on reaction pathways and identify strategies to steer the reaction toward either depolymerization or oxyfunctionalization of the dimers without interunit bond cleavage. The enzymes also target Cα-oxidized model dimers, albeit at lower rates. The findings of this study demonstrate the potential of using fungal o-methoxyphenolases for catalyzing selective ortho-hydroxylation and two-electron oxidation of lignin components and provide a new foundation for developing enzyme-based lignin valorization strategies.

Bacteria can adapt to their environment through changes in their genetic material. A large proportion of gut bacteria are shaped by host-specific diet, including complex carbohydrates. The bacterial abundance, genetic content within the same bacterial species, and sequence-level variation in genes encoding similar carbohydrate-processing enzymes may therefore vary across hosts with different diets. We previously found that the abundance of diet-degrading genes varies between hominid host populations from Tanzania. We therefore hypothesized that, in addition to these abundance differences, selective pressure could act on individual gene sequences. Here, we investigated Tanzanian hominid gut microbiome differences at the taxonomic, genetic, structural, and functional levels. We analyzed 15,146 metagenome-assembled genomes (MAGs) spanning 1563 species and identified one species with striking host-associated separation. In particular, sequence variation in a xyloglucanase-encoding gene correlated strongly with the host population. This gene was highly conserved in the Hadza population, suggesting a role in the processing of diet-associated polysaccharides. Sequence differences and structural modeling revealed amino acid substitutions near the catalytic site, and biochemical assays using xyloglucan showed that representative variants differed in activity under identical assay conditions. Collectively, our findings suggest that host lifestyle and diet contribute to population-associated sequence variation in genes encoding enzymes involved in degrading polysaccharides.

Fungal biotechnology is crucial for generating high-value enzymes and fermentation products. Despite its industrial importance, major knowledge gaps in understanding fungal genomic variation, phenotypic diversity, and protein function prediction constrain biological innovation. While advancements in sequencing technologies have established data science as an integral component in driving developments in industrial fungal biotechnology, the inherent complexity of fungal genomes and incompatible repositories continue to limit comprehensive characterization of biological relationships and their translation into industrial applications. This review examines recent progress in non-graph methodologies applied to fungal biology. Genome annotation tools uncover genetic variation through homology-based approaches and enable functional annotation of sequence variants. Metric-based methods identify horizontal gene transfer events, while multivariate techniques characterize phenotypic variation across conditions. However, the increasing diversity, scale, and multimodal nature of fungal datasets require more integrative frameworks. Graph data science, a multivariate approach to model complex relationships as networks, offers opportunities to overcome these challenges. We discuss how graph-based methods enhance the detection of genomic structural variation and enable the modeling of molecular interactions. Furthermore, we outline how these approaches facilitate the exploration of complex fungal systems through multi-taxon, reference-free analyses, that integrate evolutionary signals, functional associations, and curated knowledgebases. By surveying available fungal resources and their taxonomic and ecological representations, we identify well-characterized genera, highlight underexplored taxa requiring further data generation, and pinpoint the ecological biases inherent in current sequencing efforts. Collectively, these advancements demonstrate how graph data science can accelerate fungal research and bridge fundamental discoveries and biotechnological applications.

Aspergillus oryzae is used in solid-state fermentation (SSF) to produce plant-based foods. To this end, the substrate is inoculated with spores of this fungus. So far, the effect of inoculum size on SSF with A. oryzae has primarily focused on the production of specific enzymes. Therefore, the aim of this study was to examine the impact of inoculum size on the full secretome, combined with enzyme activity assays, assessment of colonization, substrate degradation, and sporulation. To this end, A. oryzae was grown for 7 days on whole yellow pea (Pisum sativum). Fluorescence microscopy with a GFP-expressing A. oryzae strain showed that peas had been colonized externally and internally, irrespective of inoculum size. Yet, the highest inoculum size resulted in a stronger pea biomass reduction when compared to the lowest inoculum size. By contrast, sporulation decreased with increasing inoculum size. Notably, proteomics revealed no effect of inoculum size on the protein profiles of aqueous extracts of the colonized peas. Amylases and proteases were the most abundant secreted proteins, which was consistent with their high activity in the aqueous extracts. Proteomics also identified β-1,3-glucanases and chitinases, indicating hyphal lysis. Indeed, 10-19% of the fungal proteins detected in the aqueous extracts lacked signal peptides. These data contribute to our understanding of colonization of substrates by A. oryzae and may be used to optimize SSF with this food grade fungus.

This study examines how seven microbial alpha-amylases selected from different glycoside hydrolase 13 (GH13) subfamilies (GH13_1, GH13_5, GH13_37, and GH13_42) affect the molecular and physical properties of potato starch with respect to gelation and gel properties. The results revealed distinct degradation profiles, reflecting different preferences for amylose and amylopectin. Rheological analysis of the starch gels revealed that, notably, starch treated with Um-alpha Amy, a GH13_37 alpha-amylase from an uncultured marine bacterium, had superior gel properties, while starch treated with catalytically efficient Bacillus-derived alpha-amylases of GH13_5 exhibited particularly poor gelling abilities. The results suggest that the strong gel properties of Um-alpha Amy treated starch are likely associated with a preferential, yet controlled, amylose degradation combined with a limited activity on amylopectin. Assessment of the enzyme structure models indicated a possible correlation between active site conformation and starch degradation profiles, with open conformations potentially enabling enhanced amylopectin degrading ability, and thus, poor gelling ability of the resulting starch. Furthermore, the limited thermal stability of Um-alpha Amy turned out to be a desirable trait, facilitating a more controlled enzymatic starch modification process. Altogether, these findings provide a novel insights into the significance of alpha-amylase phylogeny and classification in controlled enzymatic starch modification and highlight the potential of selected alpha-amylases for enzymatic production of modified potato starch with distinct gel properties.

In this study, 20 α-amylases covering several glycoside hydrolase 13 (GH13) subfamilies were characterized with regard to their impact on the molecular properties of gelatinized potato starch. Using a multi-analytical approach, the enzyme-induced changes in the relative amylose content, molecular weight distribution, chain length distribution, and vibrational properties (FTIR) of the starch were assessed. The results showed that structurally and phylogenetically diverse α-amylases produced distinct starch degradation patterns, resulting in different molecular properties of the starch. Principal component analysis (PCA) of the starch molecular weight and chain length distribution data enabled functional classification of the enzymes into six distinct groups, revealing amylopectin-degrading ability as a central performance parameter. While subfamily classification alone turned out to be a less reliable predictor of the α-amylases' starch degradation patterns, protein structural features - particularly active site openness - were found to correlate well with enzymatic substrate preference and proposed mode-of-action. The study highlights the benefits of a multi-analytical framework for differentiating functional nuances of α-amylases beyond conventional classification schemes, potentially providing a basis for rational selection of these for targeted starch modification in the future.

Carbohydrate esterases (CEs) catalyze the selective removal of ester-linked substituents from complex polysaccharides, influencing biomass bioprocessing. Predicting CE substrate specificity remains challenging due to the functional diversity and limited experimental characterization of CEs. Classic full sequence-based alignment approaches often fail to capture functional nuances across distant homologs. Here, we introduce a reverse prediction framework that leverages genomic context, specifically polysaccharide utilization loci (PULs), to infer natural polysaccharide targets of CE families. By integrating motif-based functional groups with large-scale co-occurrence analysis across Bacteroidota genomes, we identify substrate preferences at the clade level for 20 ce families. Subdivision of families into clades mitigated any polyspecificity observed when families were treated as a whole, highlighted unexplored regions within CE1, CE2, CE3, CE6, CE7, CE14, CE15, CE19, CE20, and partly within CE8 and CE12, and expanded functional coverage by up to 50

Abstract The combination of synthetic biology and additive manufacturing has driven major changes in production of biomaterials, especially through the use of three-dimensional (3D) bioprinting to create engineered living materials. However, current fabrication methods can be limited by prohibitive hardware costs and the inability to maintain structural fidelity in complex, free-form living architectures. This work demonstrates how to build a low-cost, open-source 3D bioprinting platform that can make complicated bacterial structures with complex geometry and high dimensional accuracy. A commercially available, conventional fused deposition modeling 3D printer was modified to create a bioprinting system that is simple to build. The modified bioprinter, which costs around $450, is less expensive than many commercial bioprinters. This 3D-printing technology uses slurry-based support bath methods featuring low-cost gelatin and agarose microparticles, resulting in structures with a high aspect ratio (>8:1) and feature sizes as small as 260 μm. The optimization of critical printing settings, including the ability of the bio-ink to retract during nonprint movements, resulted in a reduction of unwanted bacterial deposition by nearly 2 orders of magnitude. Long-term viability experiments showed that bacteria in the bioprints could survive for at least 28 days with nutrient supplementation. Additionally, 3D-printed engineered biofilms revealed that incubation conditions and extracellular matrix composition significantly impacted the mechanical properties of printed constructs, with trade-offs between matrix production and mechanical integrity. This study showcases an accessible 3D bioprinting platform for advanced bioprinting technologies, enabling development of engineered living materials with potential applications in synthetic biology, biotechnology, and tissue engineering.

Seaweeds (macroalgae) are an attractive resource for diverse microbial- and enzymatic production processes. They are abundant, underutilized, cheap, and rich in carbohydrates, and therefore have the potential to be used as a source of mono- or oligosaccharides, and as substrates for industrial fermentation processes. Many seaweed polysaccharides, including the sulfated polysaccharides ulvan and fucoidan, are however complex and heterogenous in structure, and there are currently few enzymes available to modify them, and understanding of their enzymatic depolymerization remains limited. The present study aimed to identify and characterize robust fucoidanases and ulvan lyases. Metagenomes were obtained from microbial enrichments from an intertidal hot-spring, genes identified that expressed putative fucoidanases and ulvan lyases, and following gene cloning and expression, the respective enzymes were screened for enzymatic activity. Consistent with their origin, the identified protein sequences were considerably divergent from previously characterized enzymes, with a 44 % average maximal sequence identity. In total, the study resulted in the characterization of 10 new fucoidanases (GH107 and GH168 families) and 8 new ulvan lyases (PL24, PL25 and PL40 families). Notably, the new fucoidanases appeared to have functional specificity towards fucoidan containing α-1,3 linked L-fucosyl and several functioned at high temperature. The study contributes a metagenomics-based approach to identify new seaweed polysaccharide degrading enzymes and an increased understanding of the diversity of such enzymes, which may have implications for the realization of biotechnology based valorization of seaweed biomass.

Precision fermentation of animal food proteins is an area of intense research as the requirements for more sustainable production increase. Despite significant strain engineering advances, the optimization of cell factories, including existing industrial fungal hosts, to achieve cost-effective protein yields is a major challenge. It is envisaged that the cost point of precision fermentation proteins needs to reach 10 USD/kg by 2025 and 1 USD/kg by 2035. This is a gigantic challenge to obtain high protein titers through strain and process development. A major challenge is the cost of carbon source, and improving the yield is therefore crucial for the economic feasibility. In this study, the influence of pH, temperature, and carbon-input rate was examined in a design of experiment approach to identify optimal fermentation parameters for the production of bovine beta-lactoglobulin by an industrial Aspergillus oryzae production strain. Carbon-input is a measure of carbon dosing relative to a benchmark feeding profile. The carbon-input was defined as the feed concentration multiplied by the feed-rate. This approach was used to show the relevance of the study and without compromising the confidentiality of the specific feed data. The optimal production yield, representing a protein production increase of almost 80% and a carbon yield above 70%, was achieved at pH 6.9, 36 degrees C, and a carbon-input of 1.23x. The carbon-input correlated with biomass formation and significantly affected carbon yield. These results represent a significant step toward cost reduction and implementation of feasible precision fermentation for sustainable production of alternative food protein for a growing population. The production strain used in this study has been used to produce registration batches for a current Generally Regarded As Safe notice submission to the Food and Drug Administration.

The lacto-N-biosidase (EC 3.2.1.140) LnbX from Bifidobacterium longum subsp. longum JCM 1217, a member of the glycoside hydrolase family 136 (GH136), was used for single-step lacto-N-biose (LNB) elongation of human milk oligosaccharides (HMOs) via disaccharide transglycosylation to expand the portfolio of HMOs available from enzymatic synthesis. We used the commercially available HMOs lacto-N-tetraose (LNT) as donor and lacto-N-neotetraose (LNnT) as acceptor for the synthesis of para-lacto-N-hexaose (pLNH). To improve the transglycosylation performance of LnbX (18 % molar yield), we designed 14 single mutants using two different strategies: 1) conservative substitution of conserved residues in the active site, and 2) shielding of the active site by large, hydrophobic residues. Protein engineering improved pLNH yield 1.5-fold as compared to the wild type (to 27 %). More than a 3-fold increase was obtained when optimizing the reaction conditions using the best variant, LnbX D416N, by an experimental design including reaction temperature, pH, donor substrate concentration, acceptor-to-donor (A/D) ratio, and enzyme concentration. Higher LNT concentrations and A/D ratios led to increased pLNH yields, and a high A/D ratio also increased the proportion of pLNH among the reaction products. The maximum molar yield of 57 % was obtained after 3 h of reaction at 100 mM LNT, 500 mM LNnT, 1 μM enzyme, 35 °C and pH 6.5. The LnbX D416N enzyme, which can use lacto-N-fucopentaose III (LNFP-III) as an alternative acceptor substrate, is active and stable at industrially relevant ranges of pH and temperature. The enzyme may thus be useful for diversification of the industrially available HMO portfolio.

In this study, the coupled immobilization on nanosilica of xylose dehydrogenase and alcohol dehydrogenase was accomplished with high efficiency, which was 90 %. Moreover, immobilization of the cofactors oxidized and reduced nicotinamide adenine dinucleotide, i.e., NAD+ and NADH, on silica material was examined and the impact on the effectiveness of the process was determined. The highest efficiency of NAD+ immobilization was found to be 56 %, which was obtained after 24 h of immobilization at 30 degrees C, pH 7 For NADH, the best immobilization efficiency was 53 % which was achieved after 24 h at 25 degrees C, pH 7. The KM and Vmax values were determined for various configurations of the biocatalytic systems showing, as expected, that immobilization of the enzymes decreased the catalytic rate (Vmax) and slightly increased the KM, but verifying that the immobilization of the cofactors did not significantly affect the kinetics, but would enable high conversion, and potentially continued enzymatic reaction. The use of the system configuration with co-immobilized enzymes, immobilized NAD+ and immobilized NADH thus allowed for obtaining over 90 % efficiency of xylose conversion in one batch, which was significantly higher than the systems with single free or only one immobilized cofactor. Using UV-Vis measurements, it was confirmed that effective cofactor regeneration occurred in the systems with immobilized components thus allowing for sustained enzyme catalyzed upgrading of xylose to xylonic acid.