Alginates are a family of linear anionic polysaccharides produced by brown algae and some bacteria and consist of 1 → 4 linked β-d-mannuronic acid (M), and its C5-epimer α-l-guluronic acid (G). Research has shown that oligoguluronates possess biofilm disruption properties, which combined with their high water-solubility and low toxicity, represent ideal candidates for creating novel bifunctional antifungal compounds. The progressive increase in antifungal resistance is a major global human health threat. This intrinsic resistance illustrates the need to develop improved antifungal drugs with greater efficacy against antifungal growth, while also reducing the potential for drug toxicity, resistance and tolerance. Amphotericin B is a polyene macrolide antifungal with broad specificity but has poor water solubility and is limited by infusion-related reactions and nephrotoxicity. In this work, we developed two types of Alginate-Amphotericin B conjugates with low molecular weight oligoguluronates conjugated through the reducing end. Conjugation to amphotericin B was through the carboxylic acid group or the amine group. The Alginate-Amphotericin B conjugates have reduced toxicity towards liver cells (HepG2) and kidney cells (LLC-PK1) and highly improved solubility characteristics, while maintaining anti-fungal activity.

IntroductionChitin is a highly abundant polysaccharide in nature and is linked to immune recognition of fungal infections and asthma in humans. Ubiquitous in fungi and insects, chitin is absent inmammals and plants and, thus, represents a microbeassociatedmolecular pattern (MAMP). However, highly polymeric chitin is insoluble, which potentially hampers recognition by host immune sensors. In plants, secreted chitinases degrade polymeric chitin into diffusible oligomers, which are “fed to” innate immune receptors and co-receptors. In human and murine immune cells, a similar enzymatic activity was shown for human chitotriosidase (CHIT1), and oligomeric chitin is sensed via an innate immune receptor, Toll-like receptor (TLR) 2. However, a complete system of generating MAMPs from chitin and feeding them into a specific receptor/co-receptor-aided sensing mechanism has remained unknown in mammals.MethodsThe effect of the secreted chitinolytic host enzyme, CHIT1, on the TLR2 activity of polymeric chitin preparations from shrimps, house dust mites and the fungal pathogen Candida albicans was assessed in vitro using cell lines and primary immune cells. Moreover, the regulation of CHIT1 was analyzed.ResultsHere, we show that CHIT1 converts inert polymeric chitin into diffusible oligomers that can be sensed by TLR1/TLR2 co-receptor/receptor heterodimers, a process promoted by the lipopolysaccharide binding protein (LBP) and CD14. Furthermore, we observed that Chit1 is induced via the b-glucan receptor Dectin-1 upon direct contact of immortalized human macrophages to the fungal pathogen Candida albicans, whereas the defined fungal secreted aspartyl proteases, Sap2 and Sap6, from C. albicans were able to degrade CHIT1 in vitro.DiscussionOur study shows the existence of an inducible system of MAMP generation in the human host that enables contact-independent immune activation by diffusible MAMP ligands with a striking similarity to the plant kingdom. Moreover, this study highlights CHIT1 as a potential therapeutic target for TLR2-mediated inflammatory processes that are fueled by oligomeric chitin.

ABSTRACT Broiler processing environments are a source of spoilage bacteria and potential pathogens. The aim of this study was to investigate the impact of cleaning and disinfection (C&D) on bacterial load and diversity in two broiler processing plants and to determine the efficacy of industry-standard disinfectants (DIs). C&D significantly reduced average bacterial loads on surfaces from 3.7 to 1.8 log CFU/cm 2 in Plant A and from 7.0 to 3.8 log CFU/cm 2 in Plant B ( P < 0.001). Metataxonomics revealed that Acinetobacter and an unknown Enterobacteriaceae genus dominated before C&D in Plants A and B, respectively, while Pseudomonas was predominant after C&D in both plants. Bacterial diversity only declined significantly after C&D in Plant B. Bacterial loads also declined across hygienic zones along the broiler processing line in Plant A. During slaughter, Staphylococcus , Lactobacillus , and Anoxybacillus dominated, and bacterial loads were significantly higher compared with post-slaughter zones, which were dominated by Pseudomonas . Culture-dependent analyses confirmed the presence of Pseudomonas spp. and also bacteria affecting food safety, including Listeria monocytogenes , Escherichia coli , Yersinia enterocolitica , Acinetobacter baumannii , and Pseudomonas aeruginosa . A selection of these was tested against DIs using robotic high-throughput screening. At the recommended user concentrations, DIs effectively inhibited planktonic bacteria and significantly reduced mono-species biofilms. However, none of the DIs completely eradicated all biofilms at these concentrations, with survival rates ranging from 7% to 53%, depending on the DI. In conclusion, C&D effectively reduces the bacterial burden and reshapes the bacterial microbiota with incomplete biofilm eradication by commercial DIs. IMPORTANCE Broiler meat continues to be involved in bacterial disease outbreaks. The surface microbiota in broiler processing environments can be a source of contaminating bacteria. Our study highlights the importance of effective C&D routines since potential pathogens and spoilage bacteria are found in these environments. Furthermore, the study provides evidence of biofilms surviving high concentrations of industry-standard DIs. This emphasizes the importance of additional measures to facilitate biofilm removal, such as mechanical cleaning, but also suggests that there is a need for DIs with stronger biofilm eradication capabilities. Ultimately, it is important to understand and continuously improve the state of hygiene in broiler processing plants to mitigate the risk of foodborne disease outbreaks.

The mannuronate C-5 epimerase AlgE1 from Azotobacter vinelandii introduces long blocks of guluronate (G) into alginate. AlgE1 is an elongated enzyme consisting of six modules, of which two are catalytically active modules (A-modules). For industrial applications, G-rich alginates are sought after, and previous studies have shown that AlgE1 can be used for the valorization of both seaweed-derived and microbially produced alginates, but a complete understanding of the mode of action of AlgE1 is lacking. This study gives new data on the overall shape and conformational freedom of the AlgE1 enzyme in solution in the presence and absence of a substrate. With this basis, the questions of how the modules of AlgE1 work together and how the enzyme moves on the substrate have been addressed. The two A-modules were inactivated individually, which clarified the roles of each A-module and showed that small changes in the full-length construct affect the mode of action. The relative positions of the A-modules were switched, which resulted in two new enzymes with an initial reaction rate higher than that of the WT but with a reduced capacity to form long G-blocks. To understand the orientation of AlgE1 in processing of its substrate, lyase activity was introduced at different positions in AlgE1, and it could be concluded that AlgE1 processes the substrate with the C-terminal acting first. Overall, this study gives a completely new insight into the mode of action of AlgE1, which is important for further development and use of alginate epimerases in industrial applications.

Mapping reservoirs of antimicrobial resistance (AMR) across food value chains and their environmental dissemination pathways is essential for limiting the spread and impact of AMR. The aim of this study was to investigate the prevalence of AMR genes and bacteria in sidestream materials, waste discharges, and processing environments of salmon and broiler. A targeted hybrid capture-based sequencing approach was used to characterize the resistome in samples collected from four processing plants, revealing a diverse range of AMR genes. Among these, we found several high-risk AMR genes, including the multidrug resistance genes TolC and mdtE, tetracycline genes tet(L) and tet(M), aminoglycoside genes APH(3′)-IIIa and APH(6)-Id, and beta-lactam genes mecA and mecR1. Overall, the highest numbers of AMR genes were found in samples of process wastewater and sludge, ranging from 32 to 330 unique genes. More than 300 bacterial isolates, including Enterobacterales, Enterococcus and Pseudomonas spp. were also collected and identified, and a subset was tested for antibiotic susceptibility. Antibiotic resistance among Enterococcus and Pseudomonas spp. was low. Quinolone-resistant Escherichia coli (QREC) were detected in waste discharges from two broiler processing plants, while multidrug resistant (MDR) E. coli were found only in one plant. Whole genome sequencing of MDR isolates revealed multiple plasmids and AMR genes such as sul2, ant(3″)-Ia, qnrS1, and blaCTX-M-1. Our study highlights that wastewater from food industries can contribute to the release of AMR bacteria and genes to the environment. While the prevalence of AMR bacteria in sidestream materials was low among the isolates in our collection, numerous AMR genes were detected, which may be re-introduced to new production systems.

The alginate-degrading enzyme BoPL38 of the human gut bacterium Bacteroides ovatus CP926 degrades all three polysaccharide structures found in alginate, a major constituent of brown macroalgae, making it a valuable tool for the selective production of alginate oligosaccharides with industrial and biotechnological potential. Despite its abundance, alginate's heterogeneous composition limits its full utilization. Modification by epimerases and lyases can help to overcome this limitation, but typically requires distinct enzymes for each polysaccharide structure. Here, we combined experimental and computational approaches to elucidate the catalytic machinery that enables BoPL38 to act across all alginate structures. We resolved in crystallo complexes of BoPL38 with alginate oligosaccharides, providing key insights into substrate binding. These structures informed QM/MM MD simulations, which uncovered distinct conformational and reaction pathways for mannuronate and guluronate conversion. The simulations identified different transition states, showing how a single active site architecture facilitates C5 proton abstraction at subsite +1 by Y298 and H243, enabling syn- and anti-β-elimination, respectively. A well-defined residue network mediates substrate recognition, and site-directed mutagenesis revealed that disruption of this network destabilizes the active site architecture. Notably, R292 plays a critical role in distorting the sugar at subsite +1 into a preactivated conformation while also stabilizing the active site tunnel through a salt bridge. Finally, NMR spectroscopy revealed that BoPL38 also catalyzes mannuronate-to-guluronate epimerization, highlighting its multifunctionality. These findings provide molecular insight into how a single enzyme accommodates alginate's structural diversity and offer new opportunities for enzymatic polysaccharide engineering.

Alginates are valued in many industries, due to their versatile properties. These polysaccharides originate from brown algae (Phaeophyceae) and some bacteria of the Azotobacter and Pseudomonas genera, consisting of 1 -> 4 linked beta-D-mannuronic acid (M), and its C5-epimer alpha-L-guluronic acid (G). Several applications rely on a high Gcontent, which confers good gelling properties. Because of its high natural G-content (FG = 0.60-0.75), the alginate from Laminaria hyperborea (LH) has sustained a thriving industry in Norway. Alginates from other sources can be upgraded with mannuronan C-5 epimerases that convert M to G, and this has been demonstrated in many studies, but not applied in the seaweed industry. The present study demonstrates epimerisation directly in the process of alginate extraction from cultivated Saccharina latissima (SL) and Alaria esculenta (AE), and the lamina of LH. Unlike conventional epimerisation, which comprises multiple steps, this in-process protocol can decrease the time and costs necessary for alginate upgrading. In-process epimerisation with AlgE1 enzyme enhanced G-content and hydrogel strength in all examined species, with the greatest effect on SL (FG from 0.44 to 0.76, hydrogel Young's modulus from 22 to 34 kPa). As proof of concept, an upscaled in-process epimerisation of alginate from fresh SL was successfully demonstrated.

Oligosaccharides from uronic acid-containing polysaccharides can be produced either by chemical or enzymatic degradation. The benefit of using enzymes, called lyases, is their high specificity for various glycosidic linkages. Lyases cleave the polysaccharide chain by an β-elimination reaction, yielding oligosaccharides with an unsaturated sugar (4-deoxy-l-erythro-hex-4-enepyranosyluronate) at the non-reducing end. In this work we have systematically studied acid degradation of unsaturated uronic acid oligosaccharides. Based on these findings, a method for preparing saturated oligosaccharides by enzymatic degradation of uronic acid-containing polysaccharides was developed. This results in oligosaccharides with a pre-defined distribution and proportion of sugar residues compared to the products of chemical degradation, while maintaining the chemical structure of the non-reducing end. The described method was demonstrated for generating saturated oligosaccharides of alginate, heparin and polygalacturonic acid. In the case of alginate, the ratio of hydrolysis rate of Δ-G and Δ-M linkages to that of G-G and M-M linkages, respectively, was found to be approximately 65 and 43, at pH* 3.4, 90 °C. Finally, this method has been demonstrated to be superior in the production of α-l-guluronate oligosaccharides with a lower content of β-d-mannuronate residues compared to what can be achieved using chemical depolymerization alone.

Salmon aquaculture is the fastest growing food production system in the world. Deficiencies in the quality or safety of salmon can have global repercussions. Controlling food safety aspects during production is therefore essential. Here, we investigate the state of hygiene in a salmon processing plant using next generation sequencing and classical culture-dependent methods to characterize the surface microbiota before and after cleaning and disinfection (C&D) at ten surface sampling points. Total aerobic counts revealed an average reduction in the bacterial loads of 1.1 log CFU/cm2 by C&D. The highest relative abundance in the core microbiota before C&D was assigned to Acinetobacter, Mycoplasmataceae, Pseudomonas and Enterobacteriaceae in descending order. After C&D, we observed a significant increase in the relative abundance of Pseudomonas (p < 0.05). However, variations were found between conveyors, processing machines and drains. To assess the efficacy of commercial disinfectants, we performed susceptibility assays using advanced robotic high-throughput technologies and included foodborne bacteria which may affect food safety and spoilage. These included 128 Pseudomonas isolates, 46 Aeromonas isolates and 59 Enterobacterales isolates sampled from the salmon processing plant. Generally, minimum inhibitory concentrations (MICs) of the disinfectants were below the user concentration recommended by the producer for most isolates. BacTiter-Glo biofilm assays revealed that 30 min exposure to six out of eight commercial disinfectants resulted in an average reduction of relative luminescence >95 % in 59 single-species biofilms selected for screening. However, disinfection alone may not always be sufficient to eradicate biofilms completely. C&D routines must therefore be continuously assessed to maintain food safety and quality. The results from this study can contribute to understand and improve the state of hygiene in salmon processing environments.

Airway mucus is a major barrier to the delivery of lipid-based nanoparticles in chronic airway diseases such as cystic fibrosis (CF). Receptor-Targeted Nanocomplexes (RTN), comprise mixtures of cationic lipids and bifunctional peptides with receptor-targeting and nucleic acid packaging properties. The aim of this study is to improve the mucus-penetrating properties of cationic siRNA and mRNA RTNs by combining them with low molecular weight alginate oligosaccharides, OligoG and OligoM. Cationic RTNs formulated with either alginate become strongly anionic, while PEGylated messenger RNA (mRNA) and short interfering RNA (siRNA) RTNs remain cationic. Both alginates enhance mucus diffusion rates of cationic siRNA and mRNA RTNs in a static mucus barrier diffusion model, with OligoG particularly effective. PEGylation also enhance mucus diffusion rates of siRNA RTNs but not mRNA RTNs. Electron microscopy shows that RTNs remained intact after mucosal transit. The transfection efficiency of OligoM-coated mRNA RTNs is better than those coated with OligoG or PEG, and similar to cationic RTNs. In siRNA RTN transfections, OligoM is better than OligoG although 1% PEG is slightly better than both. The combination of cationic RTNs and alginate oligosaccharides represents a promising alternative to PEGylation for epithelial delivery of genetic therapies across the mucus barrier while retaining transfection efficiency.

The alginate-degrading enzyme BoPL38 of the human gut bacterium Bacteroides ovatus CP926 degrades the three polysaccharide structures found in alginate, a major constituent of brown macroalgae with numerous industrial applications. However, the detailed mechanisms of alginate-degrading enzymes remain unclear. Crystal structures of BoPL38 complexes with alginate oligosaccharides, now shed light on the enzyme's catalytic machinery. QM/MM simulations reveal distinct conformational and reaction pathways, highlighting different transition states for mannuronate and guluronate conversion. C5 proton abstraction at subsite +1 by Y298 and H243 facilitates syn- and anti-beta-elimination reactions, respectively. Substrate recognition relies on R292 distorting the sugar at subsite +1 into a preactivated conformation, while stabilizing the active site tunnel through a salt bridge. Furthermore, NMR spectroscopy found that BoPL38 also catalyze mannuronate to guluronate epimerization in addition to its lyase function, thereby paving the way for future enzymatic alginate modification. ### Competing Interest Statement The authors have declared no competing interest.

For improved control of biomaterial property design, a better understanding of complex coacervation involving anionic polysaccharides and proteins is needed. Here, we address the initial steps in condensate formation of β-lactoglobulin A (β-LgA) with nine defined alginate oligosaccharides (AOSs) and describe their multivalent interactions in structural detail. Binding of AOSs containing four, five, or six uronic acid residues (UARs), either all mannuronate (M), all guluronate (G), or alternating M and G embodying the block structural components of alginates, was characterized by isothermal titration calorimetry, nuclear magnetic resonance spectroscopy (NMR), and molecular docking. β-LgA was highly multivalent exhibiting binding stoichiometries decreasing from five to two AOSs with increasing degree of polymerization (DP) and similar affinities in the mid micromolar range. The different AOS binding sites on β-LgA were identified by NMR chemical shift perturbation analyses and showed diverse compositions of charged, polar and hydrophobic residues. Distinct sites for the shorter AOSs merged to accommodate longer AOSs. The AOSs bound dynamically to β-LgA, as concluded from saturation transfer difference and 1 H-ligand-targeted NMR analyses. Molecular docking using Glide within the Schrödinger suite 2016-1 revealed the orientation of AOSs to only vary slightly at the preferred β-LgA binding site resulting in similar XP glide scores. The multivalency coupled with highly dynamic AOS binding with lack of confined conformations in the β-LgA complexes may help explain the first steps toward disordered β-LgA alginate coacervate structures.

Low molecular weight alginate oligosaccharides have been shown to exhibit anti-microbial activity against a range of multi-drug resistant bacteria, including Pseudomonas aeruginosa. Previous studies suggested that the disruption of calcium (Ca2+)–DNA binding within bacterial biofilms and dysregulation of quorum sensing (QS) were key factors in these observed effects. To further investigate the contribution of Ca2+ binding, G-block (OligoG) and M-block alginate oligosaccharides (OligoM) with comparable average size DPn 19 but contrasting Ca2+ binding properties were prepared. Fourier-transform infrared spectroscopy demonstrated prolonged binding of alginate oligosaccharides to the pseudomonal cell membrane even after hydrodynamic shear treatment. Molecular dynamics simulations and isothermal titration calorimetry revealed that OligoG exhibited stronger interactions with bacterial LPS than OligoM, although this difference was not mirrored by differential reductions in bacterial growth. While confocal laser scanning microscopy showed that both agents demonstrated similar dose-dependent reductions in biofilm formation, OligoG exhibited a stronger QS inhibitory effect and increased potentiation of the antibiotic azithromycin in minimum inhibitory concentration and biofilm assays. This study demonstrates that the anti-microbial effects of alginate oligosaccharides are not purely influenced by Ca2+-dependent processes but also by electrostatic interactions that are common to both G-block and M-block structures.

Alginate is a polysaccharide consisting of β-D-mannuronate (M) and α-L-guluronate (G) produced by brown algae and some bacterial species. Alginate has a wide range of industrial and pharmaceutical applications, owing mainly to its gelling and viscosifying properties. Alginates with high G content are considered more valuable since the G residues can form hydrogels with divalent cations. Alginates are modified by lyases, acetylases, and epimerases. Alginate lyases are produced by alginate-producing organisms and by organisms that use alginate as a carbon source. Acetylation protects alginate from lyases and epimerases. Following biosynthesis, alginate C-5 epimerases convert M to G residues at the polymer level. Alginate epimerases have been found in brown algae and alginate-producing bacteria, predominantly Azotobacter and Pseudomonas species. The best characterised epimerases are the extracellular family of AlgE1-7 from Azotobacter vinelandii(Av). AlgE1-7 all consist of combinations of one or two catalytic A-modules and one to seven regulatory R-modules, but even though they are sequentially and structurally similar, they create different epimerisation patterns. This makes the AlgE enzymes promising for tailoring of alginates to have the desired properties. The present review describes the current state of knowledge regarding alginate-active enzymes with focus on epimerases, characterisation of the epimerase reaction, and how alginate epimerases can be used in alginate production.

Chitin is a highly abundant polysaccharide in nature and linked to immune recognition of fungal infections and asthma in humans. Ubiquitous in fungi and insects, chitin is absent in mammals and plants and, thus, represents a microbe-associated molecular pattern (MAMP). However, the highly polymeric chitin is insoluble, which potentially hampers recognition by host immune sensors. In plants, secreted chitinases degrade polymeric chitin into diffusible oligomers, which are ‘fed to’ innate immune receptors and co-receptors. In human and murine immune cells, a similar enzymatic activity was shown for human chitotriosidase (CHIT1) and oligomeric chitin is sensed via an innate immune receptor, Toll-like receptor (TLR) 2. However, a complete system of generating MAMPs from chitin and feeding them into a specific receptor/co-receptor-aided sensing mechanism has remained unknown in mammals. Here, we show that the secreted chitinolytic host enzyme, CHIT1, converts inert polymeric chitin into diffusible oligomers that can be sensed by TLR1-TLR2 co-receptor/receptor heterodimers, a process promoted by the lipopolysaccharide binding protein (LBP) and CD14. Furthermore, we observed that Chit1 is induced via the β-glucan receptor Dectin-1 upon direct contact of immortalized human macrophages to the fungal pathogen Candida albicans , whereas the defined fungal secreted aspartyl proteases, Sap2 and Sap6, from C. albicans were able to degrade CHIT1 in vitro. Our study shows the existence of an inducible system of MAMP generation in the human host that enables contact-independent immune activation by diffusible MAMP ligands with striking similarity to the plant kingdom. Moreover, this study highlights CHIT1 as a potential therapeutic target for TLR2-mediated inflammatory processes that are fueled by oligomeric chitin.

AIMS:The aim of this study was to develop a high-throughput robotic microtiter plate-based screening assay for Candida albicans, optimizing growth conditions to replicate the filamentous biofilm growth found in vivo, and subsequently, to demonstrate the assay by evaluating the effect of nutritional drinks alone and in combination with the antifungal amphotericin B (AmB).METHODS AND RESULTS:Candida albicans cultured in a defined growth medium showed filamentous growth in microcolonies, mimicking the morphology of oral mucosal disease (oral candidiasis). Addition of nutrient drinks containing fruit juices, fish oil and whey protein to the medium resulted in changed morphology and promoted growth as free yeast cells and with weak biofilm structures. Minimum inhibitory concentration of AmB on the biofilms was 0.25 μg ml-1 , and this was eightfold reduced (0.0038 μg ml-1 ) in the presence of the nutritional drinks.CONCLUSIONS:The established assay demonstrated applicability for screening of antifungal and anti-biofilm effects of bioactive substances on C. albicans biofilm with clinically relevant morphology.SIGNIFICANCE AND IMPACT OF THE STUDY:Candida albicans is the causative agent of the majority of fungal infections globally. The filamentous morphology of C. albicans and the ability to form biofilm are traits known to increase virulence and resistance towards antifungals. This study describes the development of a plate-based in vitro screening method mimicking the filamentous morphology of C. albicans found in vivo. The assay established can thus facilitate efficient antifungal drug discovery and development.

Different types of chitosan were used to prepare membranes with enhanced antibacterial properties, via the solvent casting method.Nisin, an antimicrobial peptide, already use in food preservation, was incorporated in chitosan membranes to enhance the bactericidal effect, to obtain a starting material intended for use as wound dressings.The physico-chemical properties of the membranes were monitored and the results showed a good swelling capacity and water vapor transmission rate of the membranes.Optical characterization data showed that chitosan-based membranes could provide ultraviolet light protection while in vitro biodegradability assay demonstrated good stability of the films under enzymatic degradation.Nisin improved significantly the antibacterial effect of the membranes, while the nisin-chitosan membrane-forming solutions had a bactericidal effect against both Grampositive and Gram-negative bacteria.

Alginates are linear polysaccharides produced by brown algae and some bacteria and are composed of β-D-mannuronic acid (M) and α-L-guluronic acid (G). Alginate has numerous present and potential future applications within industrial, medical and pharmaceutical areas and G rich alginates are traditionally most valuable and frequently used due to their gelling and viscosifying properties. Mannuronan C-5 epimerases are enzymes converting M to G at the polymer level during the biosynthesis of alginate. The Azotobacter vinelandii epimerases AlgE1-AlgE7 share a common structure, containing one or two catalytic A-modules (A), and one to seven regulatory R-modules (R). Despite the structural similarity of the epimerases, they create different M-G patterns in the alginate; AlgE4 (AR) creates strictly alternating MG structures whereas AlgE1 (ARRRAR) and AlgE6 (ARRR) create predominantly G-blocks. These enzymes are therefore promising tools for producing in vitro tailor-made alginates. Efficient in vitro epimerization of alginates requires availability of recombinantly produced alginate epimerases, and for this purpose the methylotrophic yeast Hansenula polymorpha is an attractive host organism. The present study investigates whether H. polymorpha is a suitable expression system for future large-scale production of AlgE1, AlgE4, and AlgE6. H. polymorpha expression strains were constructed using synthetic genes with reduced repetitive sequences as well as optimized codon usage. High cell density cultivations revealed that the largest epimerases AlgE1 (147 kDa) and AlgE6 (90 kDa) are subject to proteolytic degradation by proteases secreted by the yeast cells. However, degradation could be controlled to a large extent either by co-expression of chaperones or by adjusting cultivation conditions. The smaller AlgE4 (58 kDa) was stable under all tested conditions. The results obtained thus point toward a future potential for using H. polymorpha in industrial production of mannuronan C-5 epimerases for in vitro tailoring of alginates.