Computer-Aided Semi-Rational Design to Enhance the Activity of l-Sorbosone Dehydrogenase from Gluconobacter oxidans WSH-004.

The titer of the microbial fermentation products can be increased by enzyme engineering. l-Sorbosone dehydrogenase (SNDH) is a key enzyme in the production of 2-keto-l-gulonic acid (2-KLG), which is the precursor of vitamin C. Enhancing the activity of SNDH may have a positive impact on 2-KLG production. In this study, a computer-aided semirational design of SNDH was conducted. Based on the analysis of SNDH's substrate pocket and multiple sequence alignment, three modification strategies were established: (1) expanding the entrance of SNDH's substrate pocket, (2) engineering the residues within the substrate pocket, and (3) enhancing the electron transfer of SNDH. Finally, mutants S453A, L460V, and E471D were obtained, whose specific activity was increased by 20, 100, and 10%, respectively. In addition, the ability of Gluconobacter oxidans WSH-004 to synthesize 2-KLG was improved by eliminating H2O2. This study provides mutant enzymes and metabolic engineering strategies for the microbial-fermentation-based production of 2-KLG.

Genome and secretome insights: unravelling the lignocellulolytic potential of Myceliophthora verrucosa for enhanced hydrolysis of lignocellulosic biomass.

Lignocellulolytic enzymes from a novel Myceliophthora verrucosa (5DR) strain was found to potentiate the efficacy of benchmark cellulase during saccharification of acid/alkali treated bagasse by ~ 2.24 fold, indicating it to be an important source of auxiliary enzymes. The De-novo sequencing and analysis of M. verrucosa genome (31.7 Mb) revealed to encode for 7989 putative genes, representing a wide array of CAZymes (366) with a high proportions of auxiliary activity (AA) genes (76). The LC/MS QTOF based secretome analysis of M. verrucosa showed high abundance of glycosyl hydrolases and AA proteins with cellobiose dehydrogenase (CDH) (AA8), being the most prominent auxiliary protein. A gene coding for lytic polysaccharide monooxygenase (LPMO) was expressed in Pichia pastoris and CDH produced by M. verrucosa culture on rice straw based solidified medium were purified and characterized. The mass spectrometry of LPMO catalyzed hydrolytic products of avicel showed the release of both C1/C4 oxidized products, indicating it to be type-3. The lignocellulolytic cocktail comprising of in-house cellulase produced by Aspergillus allahabadii strain spiked with LPMO & CDH exhibited enhanced and better hydrolysis of mild alkali deacetylated (MAD) and unwashed acid pretreated rice straw slurry (UWAP), when compared to Cellic CTec3 at high substrate loading rate.

Enhanced Electron Transfer Efficiency of Fructose Dehydrogenase onto Roll-to-Roll Thermal Stamped Laser-Patterned Reduced Graphene Oxide Films.

Herein, a strategy to stamp laser-produced reduced graphene oxide (rGO) onto flexible polymers using only office-grade tools, namely, roll-to-roll thermal stamping, is proposed, proving for the first time its effectiveness for direct bioelectrocatalysis. This straightforward, scalable, and low-cost approach allows us to overcome the limits of the integration of laser-induced rGO-films in bioanalytical devices. Laser-produced rGO has been thermally stamped (TS) onto different polymeric substrates (PET, PVC, and EVA) using a simple roll-laminator; the obtained TS-rGO films have been compared with the native rGO (untransferred) via morphochemical and electrochemical characterization. Particularly, the direct electron transfer (DET) reaction between fructose dehydrogenase (FDH) and TS-rGO transducers has been investigated, with respect to the influence of the amount of enzyme on the catalytic process. Remarkable differences have been observed among TS-rGO transducers; PET proved to be the elective substrate to support the transfer of the laser-induced rGO, allowing the preservation of the morphochemical features of the native material and returning a reduced capacitive current. Noteworthily, TS-rGOs ensure superior electrocatalysis using a very low amount of FDH units (15 mU). Eventually, TS-rGO-based third-generation complete enzymatic biosensors were fabricated via low-cost benchtop technologies. TS-rGOPET exhibited bioanalytical performances superior to the native rGO, allowing a sensitive (0.0289 µA cm-2 µM-1) and reproducible (RSD = 3%, n = 3) d-fructose determination at the nanomolar level (LOD = 0.2 µM). TS-rGO exploitability as a point-of-need device was proved via the monitoring of d-fructose during banana (Musa acuminata) postharvest ripening, returning accurate (recoveries 110-90%; relative error -13/+1%) and reproducible (RSD ≤ 7%; n = 3) data.

Surface display system of Bacillus subtilis: A promising approach for improving the stability and applications of cellobiose dehydrogenase.

Cellobiose dehydrogenase (CDH) plays a crucial role in lignocellulose degradation and bioelectrochemical industries, making it highly in demand. However, the production and purification of CDH through fungal heterologous expression methods is time-consuming, costly, and challenging. In this study, we successfully displayed Pycnoporus sanguineus CDH (psCDH) on the surface of Bacillus subtilis spores for the first time. Enzymatic characterization revealed that spore surface display enhanced the tolerance of psCDH to high temperature (80 °C) and low pH levels (3.5) compared to free psCDH. Furthermore, we found that glycerol, lactic acid, and malic acid promoted the activity of immobilized spore-displayed psCDH; glycerol has a more significant stimulating effect, increasing the activity from 16.86 ± 1.27 U/mL to 46.26 ± 3.25 U/mL. After four reuse cycles, the psCDH immobilized with spores retained 48% of its initial activity, demonstrating a substantial recovery rate. In conclusion, the spore display system, relying on cotG, enables the expression and immobilization of CDH while enhancing its resistance to adverse conditions. This system demonstrates efficient enzyme recovery and reuse. This approach provides a novel method and strategy for the immobilization and stability enhancement of CDH.

Discovery of a Novel Cellobiose Dehydrogenase from Cellulomonas palmilytica EW123 and Its Sugar Acids Production.

Cellobiose dehydrogenases (CDHs) are a group of enzymes belonging to the hemoflavoenzyme group, which are mostly found in fungi. They play an important role in the production of acid sugar. In this research, CDH annotated from the actinobacterium Cellulomonas palmilytica EW123 (CpCDH) was cloned and characterized. The CpCDH exhibited a domain architecture resembling class-I CDH found in Basidiomycota. The cytochrome c and flavin-containing dehydrogenase domains in CpCDH showed an extra-long evolutionary distance compared to fungal CDH. The amino acid sequence of CpCDH revealed conservative catalytic amino acids and a distinct flavin adenine dinucleotide region specific to CDH, setting it apart from closely related sequences. The physicochemical properties of CpCDH displayed optimal pH conditions similar to those of CDHs but differed in terms of optimal temperature. The CpCDH displayed excellent enzymatic activity at low temperatures (below 30°C), unlike other CDHs. Moreover, CpCDH showed the highest substrate specificity for disaccharides such as cellobiose and lactose, which contain a glucose molecule at the non-reducing end. The catalytic efficiency of CpCDH for cellobiose and lactose were 2.05 x 105 and 9.06 x 104 (M-1 s-1), respectively. The result from the Fourier-transform infrared spectroscopy (FT-IR) spectra confirmed the presence of cellobionic and lactobionic acids as the oxidative products of CpCDH. This study establishes CpCDH as a novel and attractive bacterial CDH, representing the first report of its kind in the Cellulomonas genus.

Amino Acid Residues Controlling Domain Interaction and Interdomain Electron Transfer in Cellobiose Dehydrogenase.

The function of cellobiose dehydrogenase (CDH) in biosensors, biofuel cells, and as a physiological redox partner of lytic polysaccharide monooxygenase (LPMO) is based on its role as an electron donor. Before donating electrons to LPMO or electrodes, an interdomain electron transfer from the catalytic FAD-containing dehydrogenase domain to the electron shuttling cytochrome domain of CDH is required. This study investigates the role of two crucial amino acids located at the dehydrogenase domain on domain interaction and interdomain electron transfer by structure-based engineering. The electron transfer kinetics of wild-type Myriococcum thermophilum CDH and its variants M309A, R698S, and M309A/R698S were analyzed by stopped-flow spectrophotometry and structural effects were studied by small-angle X-ray scattering. The data show that R698 is essential to pull the cytochrome domain close to the dehydrogenase domain and orient the heme propionate group towards the FAD, while M309 is an integral part of the electron transfer pathway - its mutation reducing the interdomain electron transfer 10-fold. Structural models and molecular dynamics simulations pinpoint the action of these two residues on the domain interaction and interdomain electron transfer.

Resolving domain positions of cellobiose dehydrogenase by small angle X-ray scattering.

The interdomain electron transfer (IET) between the catalytic flavodehydrogenase domain and the electron-transferring cytochrome domain of cellobiose dehydrogenase (CDH) plays an essential role in biocatalysis, biosensors and biofuel cells, as well as in its natural function as an auxiliary enzyme of lytic polysaccharide monooxygenase. We investigated the mobility of the cytochrome and dehydrogenase domains of CDH, which is hypothesised to limit IET in solution by small angle X-ray scattering (SAXS). CDH from Myriococcum thermophilum (syn. Crassicarpon hotsonii, syn. Thermothelomyces myriococcoides) was probed by SAXS to study the CDH mobility at different pH and in the presence of divalent cations. By comparison of the experimental SAXS data, using pair-distance distribution functions and Kratky plots, we show an increase in CDH mobility at higher pH, indicating alterations of domain mobility. To further visualise CDH movement in solution, we performed SAXS-based multistate modelling. Glycan structures present on CDH partially masked the resulting SAXS shapes, we diminished these effects by deglycosylation and studied the effect of glycoforms by modelling. The modelling shows that with increasing pH, the cytochrome domain adopts a more flexible state with significant separation from the dehydrogenase domain. On the contrary, the presence of calcium ions decreases the mobility of the cytochrome domain. Experimental SAXS data, multistate modelling and previously reported kinetic data show how pH and divalent ions impact the closed state necessary for the IET governed by the movement of the CDH cytochrome domain.

Interface engineering of cellobiose dehydrogenase improves interdomain electron transfer.

Cellobiose dehydrogenase (CDH) is a bioelectrocatalyst that enables direct electron transfer (DET) in biosensors and biofuel cells. The application of this bidomain hemoflavoenzyme for physiological glucose measurements is limited by its acidic pH optimum and slow interdomain electron transfer (IET) at pH 7.5. The reason for this rate-limiting electron transfer step is electrostatic repulsion at the interface between the catalytic dehydrogenase domain and the electron mediating cytochrome domain (CYT). We applied rational interface engineering to accelerate the IET for the pH prevailing in blood or interstitial fluid. Phylogenetic and structural analyses guided the design of 17 variants in which acidic amino acids were mutated at the CYT domain. Five mutations (G71K, D160K, Q174K, D177K, M180K) increased the pH optimum and IET rate. Structure-based analysis of the variants suggested two mechanisms explaining the improvements: electrostatic steering and stabilization of the closed state by hydrogen bonding. Combining the mutations into six combinatorial variants with up to five mutations shifted the pH optimum from 4.5 to 7.0 and increased the IET at pH 7.5 over 12-fold from 0.1 to 1.24 s-1 . While the mutants sustained a high enzymatic activity and even surpassed the IET of the wild-type enzyme, the accumulated positive charges on the CYT domain decreased DET, highlighting the importance of CYT for IET and DET. This study shows that interface engineering is an effective strategy to shift the pH optimum and improve the IET of CDH, but future work needs to maintain the DET of the CYT domain for bioelectronic applications.

Improvement of catalytic activity of sorbose dehydrogenase for deoxynivalenol degradation by rational design.

Deoxynivalenol (DON) is the most frequently contaminated mycotoxin in food and feed worldwide, causing significant economic losses and health risks. Physical and chemical detoxification methods are widely used, but they cannot efficiently and specifically remove DON. In the study, the combination of bioinformatics screening and experimental verification confirmed that sorbose dehydrogenase (SDH) can effectively convert DON to 3-keto-DON and a substance that removes four hydrogen atoms for DON. Through rational design, the Vmax of the mutants F103L and F103A were increased by 5 and 23 times, respectively. Furthermore, we identified catalytic sites W218 and D281. SDH and its mutants have broad application conditions, including temperature ranges of 10-45 °C and pH levels of 4-9. Additionally, the half-lives of F103A at 90 °C (processing temperature) and 30 °C (storage temperature) were 60.1 min and 100.5 d, respectively. These results suggest that F103A has significant potential in the detoxification application of DON.

Molecular Insights of Cellobiose Dehydrogenase Adsorption on Self-Assembled Monolayers.

Cellobiose dehydrogenase (CDH) is capable of direct electron transfer (DET) on electrodes and is a promising redox enzyme for bioelectrochemical applications. Its unique two-domain structure makes the function of CDH adsorbed on the surface of the electrode deeply affected by the external environment, such as ion species, strength, pH, and surface charge density. To date, however, the exact mechanism of how the external environment tailors the structure and dynamics of CDH adsorbed on the electrode surface still remains poorly understood. Here, multiscale simulations were performed to look for insight into the effect of Na+ and Ca2+ ions on the activation of CDH on oppositely charged self-assembled monolayer (NH2-SAM and COOH-SAM) surfaces with different surface charge densities (SCDs). Both Na+ and Ca2+ can promote CDH conformation switch from the open state to the closed state, while the promotion effect of Ca2+ is stronger than that of Na+ at the same conditions. However, the high ionic strength (IS) of Ca2+ renders the cytochrome (CYT) domain of CDH away from the NH2-SAM with low SCD. In contrast, whatever the IS, the NH2-SAM surface with high SCD can not only enhance the CYT-surface interaction but also achieve a closed-state conformation due to a similar role of Ca2+. Overall, this study gains molecular-level insights into the role of ion species and surface charge in modulating the structure and conformation of CDH on the SAM surface, thereby tailoring its activity.

Localization of Pyranose 2-Oxidase from Kitasatospora aureofaciens: A Step Closer to Elucidate a Biological Role.

Lignin degradation in fungal systems is well characterized. Recently, a potential for lignin depolymerization and modification employing similar enzymatic activities by bacteria is increasingly recognized. The presence of genes annotated as peroxidases in Actinobacteria genomes suggests that these bacteria should contain auxiliary enzymes such as flavin-dependent carbohydrate oxidoreductases. The only auxiliary activity subfamily with significantly similar representatives in bacteria is pyranose oxidase (POx). A biological role of providing H2O2 for peroxidase activation and reduction of radical degradation products suggests an extracellular localization, which has not been established. Analysis of the genomic locus of POX from Kitasatospora aureofaciens (KaPOx), which is similar to fungal POx, revealed a start codon upstream of the originally annotated one, and the additional sequence was considered a putative Tat-signal peptide by computational analysis. We expressed KaPOx including this additional upstream sequence as well as fusion constructs consisting of the additional sequence, the KaPOx mature domain and the fluorescent protein mRFP1 in Streptomyces lividans. The putative signal peptide facilitated secretion of KaPOx and the fusion protein, suggesting a natural extracellular localization and supporting a potential role in providing H2O2 and reducing radical compounds derived from lignin degradation.

A direct enzymatic evaluation platform (DEEP) to fine-tuning pyridoxal 5'-phosphate-dependent proteins for cadaverine production.

Pyridoxal 5'-phosphate (pyridoxal phosphate, PLP) is an essential cofactor for multiple enzymatic reactions in industry. However, cofactor engineering based on PLP regeneration and related to the performance of enzymes in chemical production has rarely been discussed. First, we found that MG1655 strain was sensitive to nitrogen source and relied on different amino acids, thus the biomass was significantly reduced when PLP excess in the medium. Then, the six KEIO collection strains were applied to find out the prominent gene in deoxyxylulose-5-phosphate (DXP) pathway, where pdxB was superior in controlling cell growth. Therefore, the clustered regularly interspaced short palindromic repeats interference (CRISPRi) targeted on pdxB in MG1655 was employed to establish a novel direct enzymatic evaluation platform (DEEP) as a high-throughput tool and obtained the optimal modules for incorporating of PLP to enhance the biomass and activity of PLP-dependent enzymes simultaneously. As a result, the biomass has increased by 55% using PlacI promoter driven pyridoxine 5'-phosphate oxidase (PdxH) with a trace amount of precursor. When the strains incorporated DEEP and lysine decarboxylase (CadA), the cadaverine productivity was increased 32% due to the higher expression of CadA. DEEP is not only feasible for high-throughput screening of the best chassis for PLP engineering but also practical in fine-tuning the quantity and quality of enzymes.

Silencing LINC01234 represses pancreatic cancer progression by inhibiting the malignant phenotypes of pancreatic cancer cells.

OBJECTIVE: Previous works have outlined the pivotal involvement of long intergenic non-coding RNA (lincRNA) in cancer progression, while the efficiency of LINC01234 in pancreatic cancer remained obscure. The purpose of this research is to unravel the regulatory mechanism of LINC01234 in pancreatic cancer via modulating microRNA (miR)-513a-3p and hexose 6-phosphate dehydrogenase (H6PD). METHODS: Pancreatic cancer cells were cultured and clinical tissue specimens were collected. LINC01234, miR-513a-3p and H6PD levels in pancreatic cancer cells and tissues were examined. Plasmids altering LINC01234, miR-513a-3p and H6PD expression were transfected into pancreatic cancer cells to assess the change in biological behaviors of pancreatic cancer cells. The targeting relations among LINC01234, miR-513a-3p and H6PD were validated. RESULTS: LINC01234 and H6PD levels were elevated while miR-513a-3p level was reduced in pancreatic cancer cells and tissues. LINC01234 deficiency hindered the malignant biological activities of pancreatic cancer cells. MiR-513a-3p depletion or H6PD elevation could abrogate the inhibitory effects of LINC01234 silencing on pancreatic cancer cells. LINC01234 sponged miR-513a-3p that targeted H6PD. CONCLUSION: The reduced LINC01234 exerts inhibitory impacts on pancreatic cancer cells via targeting miR-513a-3p to restrain H6PD level. The current study broadens the understanding of LINC01234 function and affords novel therapeutic targets for pancreatic cancer treatment.

Structural design of anthraquinone bridges in direct electron transfer of fructose dehydrogenase.

Multi-functional small molecules attached to an electrode surface can bind non-covalently to the redox enzyme fructose dehydrogenase (FDH) to ensure efficient electrochemical electron transfer (ET) and electrocatalysis of the enzyme in both mediated (MET) and direct (DET) ET modes. The present work investigates the potential of exploiting secondary, electrostatic and hydrophobic interactions between substituents on a small molecular bridge and the local FDH surfaces. Such interactions ensure alignment of the enzyme in an orientation favourable for both MET and DET. We have used a group of novel synthesized anthraquinones as the small molecule bridge, functionalised with electrostatically neutral, anionic, or cationic substituents. Particularly, we investigated the immobilisation of FDH on a nanoporous gold (NPG) electrode decorated with the novel synthesised anthraquinones using electrochemical methods. The best DET-capable fraction out of four anthraquinone derivatives tested is achieved for an anthraquinone functionalised with an anionic sulphonate group. Our study demonstrates, how the combination of chemical design and bioelectrochemistry can be brought to control alignment of enzymes in productive orientations on electrodes, a paradigm for thiol modified surfaces in biosensors and bioelectronics.

Engineering bio-interfaces for the direct electron transfer of Myriococcum thermophilum cellobiose dehydrogenase: Towards a mediator-less biosupercapacitor/biofuel cell hybrid.

Direct electron transfer (DET) of enzymes on electrode surfaces is highly desirable both for fundamental mechanistic studies and to achieve membrane- and mediator-less bioenergy harvesting. In this report, we describe the preparation and comprehensive structural and electrochemical characterization of a three-dimensional (3D) graphene-based carbon electrode, onto which the two-domain redox enzyme Myriococcum thermophilum cellobiose dehydrogenase (MtCDH) is immobilized. The electrode is prepared by an entirely novel method, which combines in a single step electrochemical reduction of graphene oxide (GO) and simultaneous electrodeposition of positively charged polyethylenimine (PEI), resulting in a well dispersed MtCDH surface. The resulting MtCDH bio-interface was characterized structurally in detail, optimized, and found to exhibit a DET maximum current density of 7.7 ± 0.9 µA cm-2 and a half-lifetime of 48 h for glucose oxidation, attributed to favorable MtCDH surface orientation. A dual, entirely DET-based enzymatic biofuel cell (EBFC) was constructed with a MtCDH bioanode and a Myrothecium verrucaria bilirubin oxidase (MvBOD) biocathode. The EBFC delivers a maximum power density (Pmax) of 7.6 ± 1.3 µW cm-2, an open-circuit voltage (OCV) of 0.60 V, and an operational lifetime over seven days, which exceeds most reported CDH based DET-type EBFCs. A biosupercapacitor/EBFC hybrid was also constructed and found to register maximum power densities 62 and 43 times higher than single glucose/air and lactose/air EBFCs, respectively. This hybrid also shows excellent operational stability with self-charging/discharging over at least 500 cycles.

Cellobiose dehydrogenase in biofuel cells.

Enzymatic biofuel cells utilize oxidoreductases as highly specific and highly active electrocatalysts to convert a fuel and an oxidant even in complex biological matrices like hydrolysates or physiological fluids into electric energy. The hemoflavoenzyme cellobiose dehydrogenase is investigated as a versatile bioelectrocatalyst for the anode reaction of biofuel cells, because it is robust, converts a range of different carbohydrates, and can transfer electrons to the anode by direct electron transfer or via redox mediators. The versatility of cellobiose dehydrogenase has led to the development of various electrode modifications to create biofuel cells and biosupercapacitors that are capable to power small electronic devices like biosensors and connect them wireless to a receiver.

Ensemble-based high-throughput virtual screening of natural ligands using the Super Natural-II database against cell-wall protein dTDP-4-dehydrorhamnose reductase (RmlD) in Mycobacterium tuberculosis.

The disease Tuberculosis (TB) is caused by a bacterium called Mycobacterium tuberculosis (Mtb). The bacterial cell-wall consists of peptidoglycan layer maintains the cellular integrity and cell viability. The main problem resides in the cell cycle of Mycobacterium tuberculosis in its quiescent form which is not targeted by any drugs hence there is an immediate need for new antibiotics to target the cell wall. The current study deals with the dTDP-4-dehydrorahmnose reductase (RmlD) which is the final enzyme in the series of cell-wall proteins of Mtb. The RmlD is a part of Carbohydrate biosynthesis has been considered as a good drug target for the novel class of antibiotics. Our study begins with the protein structure prediction, Homology studies were conducted using the Phyre2 web server. The structure is then refined and subjected to molecular dynamics simulations for 50 ns using GROMACS. The clustering analysis has been carried out and generated 41 clusters with 2 Å as the cut-off. Blind docking virtual screening was performed against RmlD protein using the Super Natural-II database with AutoDock4.0. its results helped to screen top ligands based on best binding energies. In both dockings, there are some common residues in which the ligands are interacting and forming the Hydrogen bonds such as Asp-105, Val-158, Thr-160, Gly-161, Arg-224, Arg-256. The ligand-567 giving the best results by being in the top-3 of all the clusters in both blind docking as well as the active-site docking. Hence ligand-567 can be a potential inhibitor of RmlD which can further inhibit the cell-wall synthesis of Mycobacterium tuberculosis.Communicated by Ramaswamy H. Sarma.

Reagentless D-Tagatose Biosensors Based on the Oriented Immobilization of Fructose Dehydrogenase onto Coated Gold Nanoparticles- or Reduced Graphene Oxide-Modified Surfaces: Application in a Prototype Bioreactor.

As electrode nanomaterials, thermally reduced graphene oxide (TRGO) and modified gold nanoparticles (AuNPs) were used to design bioelectrocatalytic systems for reliable D-tagatose monitoring in a long-acting bioreactor where the valuable sweetener D-tagatose was enzymatically produced from a dairy by-product D-galactose. For this goal D-fructose dehydrogenase (FDH) from Gluconobacter industrius immobilized on these electrode nanomaterials by forming three amperometric biosensors: AuNPs coated with 4-mercaptobenzoic acid (AuNP/4-MBA/FDH) or AuNPs coated with 4-aminothiophenol (AuNP/PATP/FDH) monolayer, and a layer of TRGO on graphite (TRGO/FDH) were created. The immobilized FDH due to changes in conformation and spatial orientation onto proposed electrode surfaces catalyzes a direct D-tagatose oxidation reaction. The highest sensitivity for D-tagatose of 0.03 ± 0.002 µA mM-1cm-2 was achieved using TRGO/FDH. The TRGO/FDH was applied in a prototype bioreactor for the quantitative evaluation of bioconversion of D-galactose into D-tagatose by L-arabinose isomerase. The correlation coefficient between two independent analyses of the bioconversion mixture: spectrophotometric and by the biosensor was 0.9974. The investigation of selectivity showed that the biosensor was not active towards D-galactose as a substrate. Operational stability of the biosensor indicated that detection of D-tagatose could be performed during six hours without loss of sensitivity.

A new carbohydrate-active oligosaccharide dehydratase is involved in the degradation of ulvan.

Marine algae catalyze half of all global photosynthetic production of carbohydrates. Owing to their fast growth rates, Ulva spp. rapidly produce substantial amounts of carbohydrate-rich biomass and represent an emerging renewable energy and carbon resource. Their major cell wall polysaccharide is the anionic carbohydrate ulvan. Here, we describe a new enzymatic degradation pathway of the marine bacterium Formosa agariphila for ulvan oligosaccharides involving unsaturated uronic acid at the nonreducing end linked to rhamnose-3-sulfate and glucuronic or iduronic acid (Δ-Rha3S-GlcA/IdoA-Rha3S). Notably, we discovered a new dehydratase (P29_PDnc) acting on the nonreducing end of ulvan oligosaccharides, i.e., GlcA/IdoA-Rha3S, forming the aforementioned unsaturated uronic acid residue. This residue represents the substrate for GH105 glycoside hydrolases, which complements the enzymatic degradation pathway including one ulvan lyase, one multimodular sulfatase, three glycoside hydrolases, and the dehydratase P29_PDnc, the latter being described for the first time. Our research thus shows that the oligosaccharide dehydratase is involved in the degradation of carboxylated polysaccharides into monosaccharides.