Simultaneously, HepG2 cells enzymatically convert 2-cyano-6-methoxybenzothiazole into 2-cyano-6-hydroxybenzothiazole that is released in the extracellular space. d-Cysteine and 2-cyano-6-hydroxybenzothiazole react to form d-luciferin. The ACs respond to this signal by converting d-luciferin into luminescence due to the presence of encapsulated luciferase in the ACs. As a result, the ACs can eavesdrop on the mammalian cells to evaluate the level of hepatic CYP1A2 function.Cellular communication is a fundamental feature to ensure the survival of cellular assemblies, such as multicellular tissue, via coordinated adaption to changes in their surroundings. Consequently, the development of integrated semi-synthetic systems consisting of artificial cells (ACs) and mammalian cells requires feedback-based interactions. Here, we illustrate that ACs can eavesdrop on HepG2 cells focusing on the activity of cytochrome P450 1A2 (CYP1A2), an enzyme from the cytochrome P450 enzyme family. Specifically,  The complexity of a living cell can be divided into its essential cellular hallmarks , such aset al. . Intereset al. , lipid-bet al. , platinutoxicity , H2O2 scgregates , Tβ4-exoal cells , or lighal cells .The next step forward requires more interactivity between the different units, i.e. there should be a feedback loop or communication. This advancement aims to mimic cellular communication, which is essential to coordinate behaviour in living multicellular assemblies and tissue to adapt to changes in the surroundings and maintain overall homeostasis.2O2 from glucose oxidase-containing giant unilamellar vesicle as an AC model to red blood cells, which in response produce resorufin, has been demonstrated [Communication has previously been demonstrated between different populations of ACs –22, or bnstrated . Furthernstrated . We havenstrated .d-cysteine, which was released upon reduction of the linking disulfide bond with either glutathione reductase (GR) or dithiothreitol (DTT). d-Cysteine reacted with 2-cyano-6-hydroxybenzothiazole (CHB), which was produced by CYP1A2 catalysis in HepG2 cells and released to the extracellular space to create d-luciferin, which was converted to a luminescence signal by luciferase encapsulated in the ACs.Herein, we demonstrated that ACs can ‘eavesdrop' on hepatocytes focusing on the expression level of cytochrome P450 1A2 (CYP1A2), an enzyme from the CYP enzyme family . Specifi. 2. 2.1N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), dimethyl sulfoxide (DMSO), N-hydroxysuccinimide (NHS), poly(l-lysine) hydrobromide , phosphate-buffered saline, 0.25% trypsin EDTA, calcium chloride dihydrate (CaCl2), adenosine 5′-triphosphate disodium salt hydrate , magnesium chloride hexahydrate (MgCl2), minimum essential medium Eagle , sodium pyruvate, 2-cyano-6-hydroxybenzothiazole , d-luciferin (L9504), luciferase from photinus pyralis (SRE0045), l-glutathione oxidized (G4376), glutathione reductase from baker's yeast , dithiothreitol (DTT), nicotinamide adenine dinucleotide phosphate , glutathione disulfide (GSSG), 5,5′-dithio-bis-(2-nitrobenzoic acid) , salicylamide, S-(2-pyridylthio)cysteamine hydrochloride, phosphate-buffered saline (PBS), clear round bottom 96-well plate ultra-low attachment, and sodium chloride were purchased from Sigma Aldrich.4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid , l-guluronate (G block)  and ultrapure high molecular weight sodium alginate with high G block content  were purchased from NovaMatrix. Fetal bovine serum (FBS), Dulbecco's Modified Eagle Medium , MEM non-essential amino acid solution, glutamine, streptomycin and penicillin, Pierce Dye Removal Columns, and DyLight 633 NHS Ester were purchased from Thermo Fisher Scientific. µ-Slide VI 0.4  was purchased from Ibidi. d-Cysteine (H62627.03), 2-cyano-6-methoxybenzothiazole , Corning cell strainers , 96-well plate (white with clear bottom or clear), calcein-AM, and qualitative filter papers grade 600 (516–0309) were purchased from VWR.Ultrapure low molecular weight sodium alginate with high α-−1 resistance) was provided by an ELGA Purelab Ultra system .HEPES buffer consisted of 10 mM HEPES and 150 mM NaCl at pH 7.4. Ultrapure water  is referred to as l-cys free media.DMEM supplemented with 1% . 2.2. 2.2.1H) was functionalized according to a previously established protocol by Zelikin et al. [H was conjugated with a protected cysteamine, 2-pyridylthiocysteamine, and was subsequently reacted with d-cysteine at a basic pH to allow deprotonation of the thiol group and thiol–disulfide exchange, yielding AlgCys .In total, 500 mg ultrapure high molecular weight sodium alginate with high G block content  and 37.5 mg AlgH were dissolved in 5 ml ultrapure H2O to make a 15 mg ml−1 solution of AlgL/AlgH. 37.5 mg AlgL and 37.5 mg AlgCys were dissolved in 5 ml ultarpure H2O to make a 15 mg ml−1 solution of AlgL/AlgCys. Alginate mixtures were dissolved by rotating at room temperature for at least 2 h.37.5 mg ultrapure low molecular weight sodium alginate with high G block content  in Eppendorf tubes for final DTT concentrations of 0, 0.1, 1, 2.5 or 5 mM. The samples were left overnight on a rotary shaker to allow for the reduction of the disulfide bond of AlgCys. Then, the samples were washed by adding 100 µl sample and 400 µl PBS into a 10 kDa spin filter, and the samples were centrifuged at 12 × 103 rpm for 15 min. This cleaning step was repeated 7× before the amount of free thiols present in the alginate mixtures (AlgL/AlgH or AlgL/AlgCys) and the filtrate was measured using Ellman's reagent. 5 µl thiol-containing sample, 70 µl PBS and 50 µl Ellman's reagent were added to a 96-well plate. A standard curve of d-cysteine (0–1 mM) was used to calculate the amount of free thiols. The plate was left for 2 min after the addition of Ellman's reagent to develop and the absorbance at 412 nm was measured. The amount of free thiols is plotted as the expected amount from 50 µl AlgL/AlgH or AlgL/AlgCys to make it comparable to the experiments outlined in §§2.3.2, 2.5, 2.6.3 and 2.7.4.The degree of functionalization (DoF) of Alg. 2.2.32PO4 buffer  or l-cys free media was evaluated. First, stock solutions of GR, NADPH, DTNB and GSSG were prepared. A 6 U ml−1 solution of GR was prepared from the provided stock solution and subsequently, 15 µl 6 U ml−1 GR was added to 987 µl buffer for a GR solution of 0.09 U ml−1. 16 µl of an 80 mg ml−1 NADPH solution in ddH2O was added to 1 ml buffer or l-cys free media, resulting in a 1.26 mg ml−1 NADPH solution. 87.6 µl 3 mg ml−1 DTNB in DMSO was added to 912.4 µl buffer or l-cys free media to give a 0.26 mg ml−1 DTNB solution. A 6 mg ml−1 GSSG solution in buffer or l-cys free media was prepared. The assay was performed by adding the following components in the following order to a clear 96-well plate: 25 µl NADPH (1.26 mg ml−1), 50 µl GSSG (6 mg ml−1), 50 µl DTNB (0.26 mg ml−1) and 100 µl GR (0.09 U ml−1). 100 µl l-cys free media or buffer were used in the last step as the negative control. The reaction was monitored immediately after the addition of GR by measuring the absorbance at λabs = 412 nm every 10 s for 20 min.The activity of GR in either a KH. 2.2.4d-cysteine released from the alginate mixtures AlgL/AlgH or AlgL/AlgCys was evaluated upon treatment with GR or DTT for 1 h at 37°C. CHB was added to react with released d-cysteine to form d-luciferin. 10 µl 15 mg ml−1 AlgL/AlgH or AlgL/AlgCys, 40 µl PBS, and 1 µl 5 mM CHB  were added to Eppendorf tubes. Either (1) 49 µl l-cys free media, (2) 42.6 µl l-cys free media, 3.2 µl 25 U ml−1 GR , and 3.2 µl 16 mg ml−1 NADPH , or (3) 45.8 µl l-cys free media and 3.2 µl 78 mM DTT  were added to the AlgL/AlgH or AlgL/AlgCys solutions. The Eppendorf tubes were left for 1 h at 37°C, before 50 µl was transferred to a white 96-well plate. 50 µl d-luciferin detection reagent (LDR) consisting of 0.5 mg luciferase, 10 mM MgCl2 and 120 µM ATP per ml (in PBS) was added to each well. The luminescence was read after 10 min using a PerkinElmer Enspire 2300 multilabel plate reader.The amount of . 2.3. 2.3.1−1 AlgL/AlgH or AlgL/AlgCys solutions were loaded into a 20 ml syringe and connected to the Encapsulator B-390 through a plastic tube. The syringe was fixed onto a pump for a controlled injection speed of 1.1 ml min−1 through the Encapsulator B-390 with an inner nozzle diameter of 80 µm and an outer nozzle diameter of 120 µm. A frequency of 1300 Hz, an amplitude of 3, and an 800 V voltage were used during particle fabrication. The formed alginate particles were collected in a beaker containing 0.1 M CaCl2 solution while stirring and were allowed to cross-link for approximately 5 min at room temperature. The particles were then collected in a 40 µm cell strainer followed by rinsing with PBS buffer. Particles made from AlgL/AlgH or AlgL/AlgCys solutions yielded AC0 or ACCys, respectively. The ACs were visualized by using an inverted Olympus microscope (IX81), and the diameters were measured using ImageJ software. The size distribution was determined using Origin software  and a minimum of 200 beads from three independent repeats were measured.Alginate beads were fabricated using an Encapsulator B-390 (Buchi). 5 ml of 15 mg ml. 2.3.20 and ACCys, 40 µl PBS and 1 µl 5 mM CHB were added to each well and the conditions of (1) no cleavage, (2) GR and (3) DTT were applied as outlined in §2.2.4 in the same volumes. The plate was left for 1 h at 37°C, 50 µl supernatant was transferred to a white 96-well plate, and 50 µl LDR was added to each well. The luminescence was read after 10 min using a PerkinElmer Enspire 2300 multilabel plate reader.10 mg ACd-cysteine is given as the obtained amount per 50 mg AC0 or ACCys. The statistical significance used to compare the means was determined using a one-way analysis of variance (ANOVA) followed by a Tukey's multiple comparison post hoc test.When plotting the data, the amount of obtained . 2.42 (TC-treated) culture flasks in MEME supplemented with 1% l-glutamine (2 mM), 1% MEM non-essential amino acid solution, 1% Pen Strep (100 µg ml−1 streptomycin and 100 U ml−1 penicillin) and 10% FBS (growth medium) at 37°C and 5% CO2. Experiments where ACs eavesdrop on HepG2 cells were performed in l-cys free media.The human liver cancer cell line HepG2 was purchased from European Collection of Cell Cultures. HepG2 cells were cultured in 25 cm. 2.4.13 cells per well in 100 µl media. The well plates were kept in a humidified atmosphere at 37°C and 5% CO2 for either 24 h or 48 h. Then, the medium was removed and either new medium containing 0.5% (v/v) DMSO (no induction) or medium containing either 10 µM BNF or indirubin  DMSO) was added to the wells. CYP1A2 expression was evaluated by adding the luminometric substrate CMB, which reacted with d-cysteine to form d-luciferin that subsequently was detected by the addition of luciferase.CYP1A2 expression was induced in HepG2 cells using the aryl hydrocarbon receptor (AhR) agonists β-naphthoflavone (BNF) or indirubin . HepG2 cl-cys free media supplemented with 3 mM salicylamide, 0.5 µl 5 mM CMB  and 0.5 µl 10 mM d-cysteine  to each well. The plate was left to incubate at 37°C and 5% CO2 for 1 h. 50 µl LDR was added to each well and the plate was read after 10 min. As controls, the addition of CMB, d-cysteine or ATP was omitted. A standard curve was made by preparing a dilution series of d-luciferin from 0 to 10 µM for the calculation of the amount of d-luciferin formed by the HepG2 cells.Following the induction, wells were washed 2× with PBS, and the CYP1A2 activity was assessed by adding 49 µl . 2.53 cells per µ-slide VI 0.4 (microslide). 100 µl of 30 × 104 cells ml−1 was added to one chamber while tilted approximately 20°, and 60 µl was added to the second inlet chamber. The maximum volume of each chamber is 160 µl and each microslide consisted of six sets of neighbouring chambers. The microslides were placed in the incubator at 37°C in 5% CO2 while remaining tilted and the cells were allowed to adhere and grow at this tilted angle . New medium with 0.5% (v/v) DMSO (no induction) or 1 µM indirubin (at 0.5% v/v) was added to the cells after 24 h and 48 h. The next day, the chamber was washed 2× with PBS and 91 µl l-cys free media was added. 0.7 µl 10 mM CMB  was added to each chamber. Then, ACs, fabricated the day before, were added to the second inlet chamber by mixing 20 mg AC0 or ACCys with 40 µl l-cys free media. Next, either (1) 9.0 µl l-cys free media (no cleavage), (2) 4.5 µl 25 U ml−1 GR, 4.5 µl 16 mg ml−1 NADPH or (3) 4.5 µl 78 mM DTT and 4.5 µl l-cys free media were added and the microslides were left for 2 h at 37°C and 5% CO2. Next, 50 µl medium was transferred to a white 96-well plate, 50 µl freshly prepared LDR was added to each well, and the luminescence was read after 10 min using a PerkinElmer Enspire 2300 multilabel plate reader. A standard curve was prepared by mixing 0–10 µM CHB with 300 µM d-cysteine for 2 h at 37°C and 5% CO2, followed by transferring 25 µl to a white 96-well plate, and adding 25 µl PBS and 50 µl LDR. A standard curve was prepared with each experiment to evaluate the amount of CHB formed from HepG2 cells. The statistical significance used to compare the means was determined using a one-way ANOVA followed by a Tukey's multiple comparison post hoc test.HepG2 cells were seeded at a final concentration of 30 × 10. 2.6. 2.6.1l-lysine (PLL) was conjugated with rhodamine B isothiocyanate (R) as previously described [R by mixing 1 ml 50 mg ml−1 AC solution (in HEPES buffer) with 1 ml 1.2 mg ml−1 PLLR solution (in HEPES buffer) in a 1 : 1 ratio. The solution was left to incubate for 1 h at 4°C to allow coating of the ACs with PLLR. Subsequently, the solution was washed 3× with 0.1 M CaCl2 solution by spinning the solution at 4.5 × 103 rpm for 5 min. The resulting 50 mg PLLR coated ACs were resuspended in 1 ml growth medium. The solution was diluted 5× into PBS buffer, and the concentration was determined by manually counting all beads in 3 × 2 µl of solution. The ACs were visualized by taking bright field and fluorescence microscopy images with a U-MNG filter cube (Olympus) for detecting PLLR using an inverted Olympus microscope (IX81). The diameters of a minimum of 200 beads from three independent repeats were measured on bright field microscopy images using ImageJ software, and the size distribution was determined using Origin software .ACs were fabricated as described in §2.3.1. After collection in the 40 µm cell strainer, the ACs were filtered through a 100 µm cell strainer and the ACs smaller than 100 µm were collected. Poly-escribed . The ACs. 2.6.20 or ACCys with PLLR coating were co-cultured with HepG2 cells at a cell-to-AC ratio of 32:1 in 96-well plates with an ultralow attachment surface. 20 000 HepG2 cells and 606 ACs were used per aggregate in 200 µl growth medium. A stock solution of 30.3 × 103 ACs per ml (in growth medium) was prepared and 20 µl was added to each well. A stock solution of 40 × 103 HepG2 cells per ml was prepared and 100 µl was added to each well, followed by 80 µl growth medium. The aggregates were left to form for 3 days, and the growth medium was exchanged on day 3 and 4 to growth medium with or without 1 µM indirubin . After 48 h induction, on day 5, the eavesdropping experiment was performed (see §2.6.3).ACR using an inverted Olympus microscope (IX81). Aggregates exposed to GR or DTT were imaged and compared as well as untreated aggregates. Further, aggregates treated with DTT were visualized using confocal laser scanning microscopy (CLSM) with a Zeiss LSM700 CLSM . A staining solution of 4 µM calcein-AM in PBS was prepared, and the aggregates were stained for 10 min. Then, the aggregates were transferred using a 1 ml pipette to a glass slide for imaging with CLSM using a 20× objective. Tile scans were obtained with a resolution of 512 × 512 pixels, and dimensions of 5 × 5 or 3 × 3. Cleaved calcein-AM in live cells was imaged using λex/λem = 488/493–532 nm, and PLLR was visualized using λex/λem = 555/560–800 nm.The aggregates were visualized by taking bright field and fluorescence microscopy images with a U-MNG filter cube (Olympus) for detecting PLL. 2.6.3l-cys free media was added. Next, 1 µl 10 mM CMB , and either (1) 12.8 µl l-cys free media (no cleavage), (2) 6.4 µl 25 U ml−1 GR and 6.4 µl 16 mg ml−1 NADPH, or (3) 6.4 µl 78 mM DTT and 6.4 µl l-cys free media were added and the well plate was left for 2 h at 37°C and 5% CO2. Next, 50 µl media was transferred to a white 96-well plate and 50 µl LDR was added to each well. The luminescence was read after 10 min using a plate reader. A standard curve of CHB was prepared with each experiment as outlined in §2.5. The statistical significance used to compare the means was determined using a one-way ANOVA followed by a Tukey's multiple comparison post hoc test.The eavesdropping experiment was performed after growing the AC/HepG2 aggregates for 5 days, including 2 days of induction. Then, the aggregates were washed 2× with PBS and 186.2 µl . 2.7. 2.7.1−1, pH 7.4). DyLight (5 µg ml−1 in 125 µl HEPES) was mixed 1:1 (v/v) with luciferase for a final concentration of 2.5 µg ml−1 and 2 mg ml−1 luciferase, respectively. The Eppendorf was left to rotate for 5 h at room temperature. Then, the non-conjugated dye was removed by purification on a resin fluorescent dye removal column. The column was prepared as per the manufacturer's protocol. Briefly, the dye removal resin was mixed thoroughly and 250 µl was transferred to a spin column. The column was centrifuged for 30 s at 1000×g to remove the storage solution, the collection tube discarded, and the resin column placed in a new collection tube. 250 µl of the labelling reaction solution with luciferase (2 mg ml−1) and DyLight 633 was transferred to the resin and was pipetted up and down to mix thoroughly. Then, the column was centrifuged for 30 s at 1000×g, and the purified protein  was collected as the filtrate.Luciferase was fluorescently labelled using DyLight 633 Amine-Reactive Dye. The NHS ester-activated derivative of DyLight 633 was used to label the free amines on luciferase. First, 0.5 mg of luciferase was dissolved in 125 µl HEPES  DMSO (no induction) or 1 µM indirubin (at 0.5% v/v) was added to the HepG2 cells after 24 h and 48 h. The next day, the chamber was washed 2× with PBS and 91 µl l-cys free media was added. Paper chips were added to each well using tweezers and 20 mg ACs were added to each paper chip. Next, 0.5 µl 10 mM CMB  and either (1) 6.4 µl l-cys free media (no cleavage), (2) 3.2 µl 25 U ml−1 GR and 3.2 µl 16 mg ml−1 NADPH or (3) 3.2 µl 78 mM DTT and 3.2 µl l-cys free media were added directly to the media underneath the paper chip. The well plate was left for 2 h at 37°C and 5% CO2. Next, 100 µl of a 10 mM MgCl2 and 120 µM ATP solution (in PBS) was added to each well and the luminescence was read after 10 min using a plate reader. A standard curve of CHB was prepared with each experiment as outlined in §2.5.HepG2 cells were seeded in at a final concentration of 30 × 10d-cysteine upon reduction. The statistical significance used to compare the means was determined using a one-way ANOVA followed by a Tukey's multiple comparison post hoc test.Further, wells were prepared without seeded HepG2 cells. Instead, 50 µM CHB  DMSO) was added to each well to evaluate the amount of released . 3. 3.1et al. [First, ACs capable of eavesdropping on HepG2 cells require the ability to send a signal on demand. Alginate was chosen as the structural scaffold of the ACs, since it is a naturally derived polysaccharide offering good biocompatibility. Hydrogels have recently gained interest as a structural scaffold in the fabrication of ACs since they replicate the gel-like properties of the intracellular environment in mammalian cells as reviewed recently by Allen et al. . Alginatet al. ,15,34, iet al. .d-cysteine via thiol–disulfide exchange . As a result, d-cysteine conjugated to high molecular weight alginate (AlgH) through a disulfide bond was obtained , which upon mixing 1:1 (v/v) with low molecular weight alginate yielded a solution of AlgL/AlgCys. The DoF of AlgCys was determined by treating the AlgL/AlgCys solution with varying concentrations of DTT overnight to reduce the disulfide bond. The amount of free thiols was quantified using Ellman's reagent after washing the reduced AlgL/AlgCys, which corresponded to a DoF of 5.6% on AlgCys . Importantly, the pristine alginate solution AlgL/AlgH yielded no detectable thiols, and no excess DTT was found in the filtrate. Further, when treating AlgL/AlgCys with 2.5 mM DTT for 1 h, the amount of d-cysteine in the filtrate was measured by incubation with CHB, and the produced d-luciferin was measured by adding luciferase and ATP . Luminescence was only detected in the AlgL/AlgCys filtrate, and not from AlgL/AlgH, showing that d-cysteine was released upon treatment with DTT for 1 h.We used the carboxylic acid on alginate for functionalization by first conjugating 2-pyridylthiocysteamine before adding S. cerevisiae was used to catalyse the reduction of the disulfide bond using NADPH as an electron donor, as a more biologically relevant alternative to the small molecule DTT. The activity of GR was found to be approximately 45% lower in cell media than in K2HPO4 + EDTA buffer (pH 7.5) probably due to difference in salt composition or the presence of calcium .Next, the enzyme GR from L/AlgH and AlgL/AlgCys dissolved in l-cys free media were treated with either 0.8 U mL−1 GR or 2.5 mM DTT for 2 h at 37°C to release the d-cysteine. AlgL/AlgH and AlgL/AlgCys incubations in l-cys free media were used as controls. The released d-cysteine was incubated with CHB in excess for 30 min to produce d-luciferin, which was quantified by monitoring the signal obtained from its conversion by luciferase into luminescence. 50 µl AlgCys released approximately 25 µM and 150 µM d-cysteine when treated with GR and DTT, respectively , illustrating the expected ability of GR to release d-cysteine but with lower efficacy than DTT. AlgCys is not the natural substrate for GR, which can explain the lower efficacy of the enzyme to reduce the disulfide bond compared to the non-specific DTT. AlgL/AlgH gave no detectable signal and a small amount (approx. 5 µM) of passively released d-cysteine was detected for untreated AlgL/AlgCys.Following on, AlgL/AlgH and AlgL/AlgCys were then used to make microgels to act as ACs capable of eavesdropping on HepG2 cells. The microgels were fabricated employing either AlgL/AlgH or AlgL/AlgCys with the extrusion dripping method using an Encapsulator B390, followed by cross-linking in a calcium bath resulting in AC0 and ACCys, respectively, with diameters of approximately 90 µm . The amount of d-cysteine released from 50 mg ACCys  was found to be 20 µM and 60 µM upon exposure to GR and DTT, respectively, while no released d-cysteine was detected from AC0.Alg. 3.2The next step was to evaluate the activity of HepG2 cells towards the conversion of the compound CMB that was shown to be dominantly dependent on CYP1A2 . CYP actCys and HepG2 cells were added to two spatially separated inlet chambers on the same microslide, which were connected through a central channel to allow for exchange of molecules by diffusion. The HepG2 cells were seeded in an inlet chamber and left to attach for 24 h followed by induction with indirubin for 48 h before the addition of ACCys to the second inlet chamber. CMB and either GR or DTT were added to the chamber with the HepG2 cells for 2 h. The resulting d-luciferin was detected by addition of luciferase (a). The amount of d-luciferin was proportional to the amount of CHB produced by the HepG2 cells, since the ACs yielded d-cysteine in excess in the µM range (c). Pristine and induced HepG2 cells resulted in a significant difference in d-luciferin, with levels of approximately 20 nM and approximately 40 nM, respectively (b), demonstrating that the ACCys can eavesdrop on significant changes in the CHB production. Notably, GR and DTT usage for d-cysteine cleavage from ACCys yielded d-luciferin levels that were not significantly different, showing that both methods of cleavage yield an excess of d-cysteine. Further, the d-luciferin levels corresponded to the amount of CHB produced by HepG2 cells when d-cysteine was added externally to the solution rather than being supplied by ACCys . This aspect showed that the presence of ACCys did not affect the CYP activity, but served to eavesdrop on the HepG2 cells. Importantly, the use of AC0 yielded background levels of luminescence expected in the absence of d-cysteine. Similarly, omitting CMB and luciferase also only resulted in background luminescent signals . However, around approximately 10 nM d-luciferin was detected when ACCys were exposed to neither DTT nor GR, which likely reflected a small amount of spontaneously released d-cysteine as this level was lower than the amount of available CHB in solution.With the aim to illustrate that the ACs could eavesdrop on mammalian cells, ACciferase a. The amµM range c. Pristiectively b, demons. 3.3Cys when cultured with HepG2 cells into semi-synthetic aggregates. Here, alginate beads were fabricated as before using the extrusion dripping method with an added step of filtration through a 100 µm cell strainer to remove larger beads that would less readily integrate with the HepG2 cells likely due to their fast sedimentation to the bottom of the culture wells. This filtration step shifted the average diameter from approximately 90 µm  to approximately 45 µm  for AC0 and ACCys. The beads were then coated with rhodamine B isothiocyanate conjugated PLL (PLLR) for visualization and to render the beads positively charged to promote cell adhesion. The PLLR coating led to wrinkling of the surface of the particles, which was more pronounced for ACCys than for AC0  with no noticeable change in size . This observation was most likely due to the lower cross-linking density in ACCys, which had fewer carboxylic acid groups available since they were used for d-cysteine conjugation. The HepG2 cells and ACCys were seeded in a 1:32 ACCys:HepG2 cell ratio in ultralow adhesion surface round-bottom plates, and left to incubate for 2 days to allow for aggregate formation. Then, the aggregates were induced with indirubin for 2 days before imaging. Bright field microscopy images of the semi-synthetic aggregates revealed that the HepG2 cells formed a dense ring in the periphery and a higher degree of ACCys present in the centre of the aggregates . The semi-synthetic aggregates were imaged using CLSM after incubation with calcein-AM to visualize the live HepG2 cells (a). There were no observable differences in aggregate formation or HepG2 cell viability depending on the presence of ACCys or AC0 due to differences in surface morphology and alginate modification. 3D projection of z-stacks further showed the successful formation of semi-synthetic aggregates with HepG2 cells growing around ACCys and AC0 . Further, treatment with GR or DTT led to no apparent changes in the appearance of the semi-synthetic aggregates; in particular, DTT treatment did not decrease the fluorescence from the calcein-AM staining , suggesting no adverse effect on cell viability.The direct interaction between ACs and mammalian cells in aggregates is a first step towards the assembly of 3D multicellular functional semi-synthetic tissue. Thus, the aim was to demonstrate the eavesdropping capability of ACG2 cells a. There d-cysteine released from ACCys in the aggregates was quantified since fewer ACs were used to fabricate cell aggregates (approx. 600 ACs per aggregate) compared with the amount of ACs used for the eavesdropping in the microchannels (20 mg). The amount of d-cysteine released from the ACCys in the aggregates in the presence of excess CHB (50 µM) was approximately 61 nM and 1.53 µM when incubated with GR and DTT, respectively . This d-cysteine amount was significantly lower compared to the eavesdropping experiments in the microslides due to the low amount of ACCys present in the aggregates. Additionally, the aggregate environment might present a diffusion limitation. However, since the amount of CHB converted from HepG2 cells was the limiting step for the eavesdropping in microslides , enough d-cysteine was expected to be released from ACCys. In order to prevent any potential impact on the yield of d-luciferin under the reaction conditions used in the eavesdropping experiments (2 h at 37°C and 5% CO2), a range of concentrations of d-cysteine (10–500 nM) was mixed with 0–100 nM CHB to identify the detection limits in buffer solutions . For a given CHB concentration, luminescence read-outs were comparable when using d-cysteine concentrations of 500 nM, 100 nM, or 50 nM, with the detection limit for CHB being 2 nM. However, lower luminescence was observed at stoichiometric CHB concentrations when using 25 nM or 10 nM d-cysteine. Therefore, the approximately 61 nM d-cysteine cleaved from the ACCys in the aggregates using GR was sufficient for eavesdropping. However, lower yields would present a limitation from the ACCys in supplying sufficient d-cysteine for the eavesdropping. The d-cysteine yield could be improved by using DTT, increasing the ACCys to HepG2 cell ratio, conjugating higher amounts of d-cysteine to alginate, using higher amounts of AlgCys for the ACCys assembly, or a combination thereof.First, the amount of d-luciferin was detected by the addition of luciferase and monitoring of the luminescence (b). Similar levels of d-luciferin were produced when ACCys-containing aggregates were exposed to GR and DTT for d-cysteine cleavage, which were statistically significantly higher compared to untreated aggregates. There was a trend of higher amounts of produced d-luciferin in aggregates with induced HepG2 cells compared with pristine HepG2 cells, but the difference was not statistically significant. DTT treatment led to approximately 10 nM and 25 nM d-luciferin without and with indirubin treatment, respectively. This outcome was approximately 2× lower than when performing eavesdropping in microslides. However, the amount of CHB (based on the detected d-luciferin) obtained from induced cells, when differences in the amount of seeded cells were accounted for, was approximately 1.25 × 10−3 nM per cell and 1.3 × 10−3 nM per cell in aggregates and microslides, respectively. Therefore, comparable results were obtained between the two experimental set-ups. As expected, the use of AC0 in the aggregates yielded no detectable luminescence signal. Aggregates with untreated ACCys resulted in approximately 5 nM d-luciferin, which was approximately 2× lower than in the microslides, suggesting less ACCys being present in the former case. However, this level was higher than expected with the low amount of ACCys present in the aggregates, which might be due to the direct interaction with the HepG2 cells in the aggregates leading to an increased non-triggered d-cysteine release.Following on, the HepG2 cells in the aggregates were induced with indirubin for 2 days before CMB and GR or DTT were added for 2 h. The resulting nescence b. Simila. 3.4d-cysteine), but also receive the response (d-luciferin) and reply (produce luminescence) (figure 4a(i)). Therefore, luciferase was encapsulated in ACCys resulting in a(ii)).Finally, the goal was to demonstrate that the ACs could not only send the signal . Approximately 30 µM d-luciferin was detected for the induced HepG2 cells, while statistically significant lower amounts of d-luciferin (approx. 14 µM) were measured in the pristine HepG2 cells, illustrating that the luminescent signal reflected the CHB release by the HepG2 cells, i.e. successful eavesdropping was demonstrated (b). A trend towards a higher yield of d-luciferin when using DTT (approx. 37 µM) compared to GR (approx. 29 µM) was observed, which might be due to the diffusion barrier presented by the paper chip to GR resulting in slower or lower d-cysteine release, but the difference was not statistically significant.Second, the reciprocal eavesdropping of nstrated b. A tren. 4Taken together, eavesdropping of ACs on the CYP1A2 activity in HepG2 cells was demonstrated by detecting differences in pristine and induced HepG2 cells.The eavesdropping capability of the ACs illustrates an easily implemented approach to monitor a core hepatic function when co-cultured with hepatocytes. Using ACs instead of bulk assays offers opportunities to bring reaction molecules into close proximity to the partner units or spatio-temporal control of molecule synthesis and release, among others. The next step forward will be the implementation of ACs capable of reciprocal communication with mammalian cells with the goal to influence the behaviour of both the ACs and the living cells.


At pressures of 2 ÷ 8 GPa and temperatures up to 1600 °C, iron nitride Fe3N1+x is more stable than iron carbides. At 8 GPa and 1600 °C, the average particle size of iron nitride increased to 0.5 ÷ 1 μm, while simultaneously formed free carbon particles had the shape of graphite discs with a size of 1 ÷ 2 μm. Structural refinement of the iron nitride using the Rietveld method gave the best result for the space group P6322. The refined composition of the samples obtained from a mixture of ferrocene and melamine at 8 GPa/800 °C corresponded to Fe3N1.208, and at 8 GPa/1650 °C to Fe3N1.259. The iron nitride core–shell nanoparticles exhibited magnetic behavior. Specific magnetization at 7.5 kOe of pure Fe3N1.208 was estimated to be 70 emu/g. Compared to other methods, the high-pressure method allows easy synthesis of the iron nitride cores inside pure carbon shells and control of the particle size. And in general, pressure is a good tool for modifying the phase and chemical composition of the iron-containing cores.Nanoparticles of iron carbides and nitrides enclosed in graphite shells were obtained at 2 ÷ 8 GPa pressures and temperatures of around 800 °C from ferrocene and ferrocene–melamine mixture. The average core–shell particle size was below 60 nm. The graphite-like shells over the iron nitride cores were built of concentric graphene layers packed in a rhombohedral shape. It was found that at a pressure of 4 GPa and temperature of 800 °C, the stability of the nanoscale phases increases in a Fe During the last two decades, the nanoparticles of the “core–shell” type have become a hot topic of research due to rapidly expanding areas of their potential high-tech applications ,2,3. The3N@C nanoparticles have already shown excellent electrochemical Li-ion storage properties with high initial charge/discharge capacity [The Fecapacity . Althougcapacity . Iron cacapacity ,2. Onioncapacity Due to high demand, an active search is currently underway for methods of synthesis of core–shell nanomaterials with pure iron and its carbides. The most commonly used methods are pyrolysis, arc discharge, vapor deposition, as well as detonation technique , sonocheThe high-pressure method for the synthesis of core–shell nanoparticles is only sparsely explored. There are few documented works demonstrating the potential applicability of high pressures for the synthesis of core–shell nanoparticles with iron carbides ,14,15,16This work is dedicated to the synthesis of core–shell nanoparticles with iron carbide or nitride cores at high pressures of up to 8 GPa and temperatures around 800 °C. The synthesis of core–shell particles with iron nitride cores and carbon shells is a special task that is difficult to perform using other methods. The high-pressure method is more efficient. There are some interesting aspects to high-pressure synthesis. Under these conditions, the “solid state”  diffusio5H5)2  and melamine C3H6N6 , selected as starting materials, were mixed (70/30% by weight) in benzene medium in an agate mortar using ultrasound and then air-dried at 50 °C. The resulting mixture is henceforth designated as FerrMelam.Ferrocene Fe transmission electron microscope with an energy-dispersive X-ray spectroscopy (EDS) attachment.The G670 imaging plate Guinier camera  was used to perform X-ray phase and structure analysis, using Cu Kα1 radiation.A PHI 5500 ESCA , Chanhassen, MN, USA) spectrometer with Al Kα radiation  was used to conduct X-ray photoelectron spectroscopy (XPS). The high-resolution spectra had a resolution better than 0.65 eV. Differences in surface and volume were evaluated through surface etching using Ar+ ions.The magnetic properties of the synthesized samples were measured using a LakeShore Cryotronics-7404 magnetometer  at room temperature under magnetic fields of up to 7.5 kOe.Previous research studies ,13,14,15In this work, electron microscopy analysis showed that thermobaric treatment of ferrocene or a mixture of ferrocene/melamine results in the formation of a large number of nanoscale core–shell particles throughout the studied pressure range of 2 ÷ 8 GPa. EDS (energy-dispersive X-ray spectroscopy) analysis revealed that the cores are iron compounds surrounded by carbon shells. This transformation requires a temperature of about 800 °C and an exposure time of tens of minutes. The average size of the particles obtained at 800 °C did not exceed 60 nm .3C is formed from ferrocene at 800 °C and pressures of 2 and 4 GPa, while iron carbide Fe7C3 is formed at 8 GPa. TEM images of single Fe3C@C and Fe7C3@C nanoparticles are shown in 7C3 composition [X-ray phase analysis  showed tposition . In part3C carbide is 3, and for Fe7C3 it is 2.33. Thus, the data obtained show that when the synthesis pressure is increased from 2 ÷ 4 to 8 GPa and the synthesis temperature is below 800 °C, the atomic Fe/C ratio in the carbide decreases from 3 to 2.33. However, when the experiment is carried out at 8 GPa and a temperature of up to 1600 °C, the formation of Fe3C is observed again, in agreement with the literature data [The atomic Fe/C ratio in Feure data .On the contrary, HPHT treatment of the FerMelam mixture under similar conditions (800 °C/30 min) resulted in the formation of iron nitride cores only at all pressures in the range of 2 ÷ 8 GPa . It shouIt was found that the formation of core–shell nanoparticles from FerMelam at 800 °C was significantly dependent on the exposure time. A 30-min exposure time was found to be sufficient for the formation of such particles with dimensions of 40 ÷ 60 nm . It was TEM analysis of the layered carbon shells revealed that they can contain 5–50 layers which are flat in some places and curved in others. 2O3 was detected. The presence of another iron oxide, Fe3O4, in the surface layers of core–shell structures was also previously observed [2O3 composition was found inside the carbon shells. The crystal structure of the iron oxide γ-Fe2O3 is usually reported either in the cubic system (space group P4332) with partial Fe vacancy disorder or in the tetragonal system with full site ordering and c/a ratio ~ 3 [1212 with a = 0.8347 nm and c = 2.5042 nm . It has been shown [2O3 is more stable than the cubic one. The tetragonal phase of Fe2O3 has been previously obtained only in small amounts in the preparation of magnetically active nanocomposite aerogels [In the samples obtained at temperatures and/or exposure time insufficient for the formation of stable shells, in addition to iron nitride particles , particlobserved . In our atio ~ 3 . A tetraen shown  that theaerogels  and in taerogels .3N1+x. The general appearance of the diffraction patterns indicates that with increasing temperature the lines of these phases become narrower, and their intensity increases. This indicates the ordering of the structure and the reduction in defects, both in the carbon matrix and in the nitride. According to the results of scanning and transmission electron microscopy, the free carbon in the 1600 °C sample consisted of disc-shaped graphite particles with a diameter of about 1 ÷ 2 μm. The size of the round iron nitride particles was 0.2 ÷ 1.5 μm : P 312 − FR= 0.0913; P-31m − FR= 0.1000; P 6322 − FR= 0.0757. Thus, the best refinement parameters were obtained in the P 6322 group; the results are shown in 1.208 and for FerMelam/(8 GPa/800 °C/0.5 min + 8 GPa/1600 °C/0.3 min) it is Fe3N1.259.Full profile analysis of the X-ray diffraction data (Rietveld refinement) was used to refine the structure of iron nitrides obtained from FerMelam mixtures at 8 GPa/800 °C/30 min and 8 GPa/800 °C/0.5 min + 8 GPa/1600 °C/0.3 min. Three different nitride space group models were tested: P 312 , P-31m , and P 6XPS studies were carried out to clarify some details of the atomic composition and chemical bonding of the raw and ion-etched surface of the sample obtained from the ferrocene–melamine mixture via treatment at 4 GPa/800 °C/30 min. The survey spectra are presented in The XPS tests showed no iron on the raw surface while the oxygen content was above 3 at%. The nitrogen content remained constant before and after surface etching. Tests on the ion-etched surface showed 0.5–1.5 at%, 0.5–1.0 at%, and 0.7–1.2 at% of O, Fe, and N, respectively. As XPS is a surface-sensitive method, it can be concluded that the oxygen is preferentially adsorbed on the surface of the sample; the iron  is somehFor the N1s peak of nitrogen, the signal-to-noise ratio was low . HoweverxN (2 < x < 4) compound, according to [The iron Fe2p3/2 peak (approximately 707.5 eV) in rding to . If thisThus, the XPS results evidence that the iron is chemically bound to nitrogen, the carbon is in a graphite-like form and the total nitrogen content in the carbon matrix is about 0.7 ÷ 1.2 at%.5H5)2 + 30 wt%C3H6N6 mixture is more than three times higher than the nitrogen content (C/N > 3), this indicates that under the investigated conditions iron nitrides are more stable than iron carbides in core–shell nanoparticles.The experiments showed that only iron nitrides are formed from a mixture of ferrocene and melamine in the whole selected range of pressures (2 ÷ 8 GPa) and temperatures (up to ~1600 °C). Considering that the atomic carbon content in the initial 70 wt%Fe2 + 30 wt%C3H6N6 (atomic ratio N/C ~ 0.32), its concentration in the final carbon matrix is significantly lower. An estimate for the sample FerMelam/4 GPa/800 °C/30 min gives a value of 0.7 ÷ 1.2 at% N. At the same time, the chemical bonding of the residual nitrogen atoms in the carbon graphite matrix is the same as in pyridine, meaning that it could be inherited from the original melamine molecules.It was found that the approximate composition of the iron nitride core particles Fend 8 GPa b. Neithe4N (N = 0.20 atoms). The stroke in the gamma phase designation means that nitrogen atoms in Fe4N occupy an ordered position. The epsilon ε-phase of iron nitride with an approximate composition of Fe3N has a wide range of homogeneity from 15 to at least 33 at%. Increasing the nitrogen content by more than 20 at% stabilizes this nitride with a hexagonal lattice. It is also known that non-nanoscale cubic phases of iron nitride are less stable than hexagonal ones at elevated pressure and temperatures [Modern views on the equilibrium phases in the Fe-N system can be found in ,33,34. Teratures ,36,37,387C3 > Fe3C > Fe3N1+x series and at 8 GPa in the Fe3C > Fe7C3 > Fe3N1+x series. Nevertheless, it should be noted that for evaluation of the relative stability of these cores in the core–shell nanoparticles at ambient pressure, a more focused investigation has to be carried out which was not within the scope of the present work.From the results presented above it can be concluded that the thermodynamic stability of nanoscale phases increases at 4 GPa in the Fe3N1+x iron nitride is more stable than any of the iron carbides, even at temperatures up to 1600 °C. This is in agreement with experimental data on iron nitrides found as inclusions in natural mined diamonds [3C at 6 ÷ 20 GPa [Our observation is valid for temperatures up to 800 °C. Moreover, at pressures of 2 ÷ 8 GPa, the Fediamonds . It is n÷ 20 GPa . This ma÷ 20 GPa . Our dat7C3@C nanoparticles synthesized at high pressure are magnetic, and testing of their biocompatibility with living cells demonstrated negative cytotoxicity [Previous studies have shown that Fetoxicity . The res3NX, its quantity in the sample needs to be estimated. A phase composition was derived from the calculation by assuming that all carbon in the original 70/30% ferrocene/melamine mixture transforms into graphite, all ferrocene iron converts to Fe3N, and some nitrogen exits the system. The calculation revealed 29.7 wt% content for Fe3N and 70.3 wt% for graphite. Estimates of the phase composition for the sample 8 GPa/800 °C/0.5 min + 8 GPa/1600 °C/0.3 min, using Rietveld refinement, indicate that it consists of 31.79 ± 0.06 wt% Fe3N1.259 and 68.21 ± 0.08 wt% graphite carbon. Thus, the specific magnetization at 7.5 kOe of the Fe3N1.208 component of the FerMelam at 4 GPa/800 °C/30 min sample can be estimated as 69 ± 7 emu/g. Although this value does not set a record [To attribute this result to pure Fea record , it stila record  as previa record .3C carbide cores were obtained from ferrocene at 2 ÷ 4 GPa, and with Fe7C3 carbide cores at 8 GPa. Core–shell nanoparticles with iron nitride cores and stoichiometry Fe3N1+x (x = 0.2 ÷ 0.3) were obtained from ferrocene/melamine mixture (7/3 by weight) at 2 ÷ 8 GPa and temperatures approximately 800 °C. The required time for the formation of relatively stable particles is in the range of tens of minutes. The average particle size does not exceed 60 nm. Fe and N atoms do not accumulate in the carbon matrix. This was confirmed by the results of X-ray photoelectron spectroscopy investigations.Core–shell nanoparticles with Fe7C3 > Fe3C > Fe3N1+x series and at 8 GPa in the Fe3C > Fe7C3 > Fe3N1+x series.If the temperature does not exceed 800 °C, the thermodynamic stability of the nanoscale phases increases at 4 GPa in the Fe3N1+x is more stable than any of the iron carbides under all the conditions considered, i.e., pressures of 2 ÷ 8 GPa and temperatures up to 1600 °C. High-pressure–high-temperature treatment of a ferrocene/melamine mixture at 1600 °C and 8 GPa yielded iron nitride Fe3N1+x particles with sizes up to 0.2–1.5 μm and free carbon consisting of rounded flat graphite particles with a diameter of about 1–2 μm.Furthermore, the iron nitride Fe322. The refined chemical composition for the sample obtained from the mixture of the ferrocene and melamine (70/30% by weight) obtained at 8 GPa/800 °C/30 min was Fe3N1.208, and for the sample obtained at (8 GPa/800 °C/0.5 min + 8 GPa/1600 °C/0.3 min)—Fe3N1.259.When the structure of iron nitride was refined using the Rietveld method, the best result was obtained for the space group P6At temperatures of about 800 °C and pressures of 2 ÷ 8 GPa, all transformations take place in the “solid phase” in the absence of the “classical” gas phase. In the Fe-C-N system, the first stage is the decomposition of hydrocarbons, followed by the segregation of iron and nitrogen atoms, and the final stage is the nucleation and growth of core–shell nanoparticles with an iron nitride core at sufficient temperature and time.The demonstrated magnetic behavior of iron nitride core–shell nanoparticles obtained from the ferrocene/melamine mixture at 4 GPa/800 °C/30 min creates the opportunity for their applications in biology and medicine, and also for the design of reusable catalysts capable of manipulation using magnets in the reaction media ,45.Thus, it has been shown that high pressures (up to 8 GPa) can provide an effective tool for the synthesis of core–shell nanoparticles, allowing control of the phase composition of cores consisting of iron compounds such as carbides and nitrides.


The first structural determination of the α-linkage-specific fucoidan hydrolase Mef1 (GH107) is reported, including the positioning of two calcium sites and the two main amino acids involved in the active-site hydrolytic mechanism. The structural determination also led to the discovery of a water wire leading from the exterior into the active site of the enzyme. Muricauda eckloniae is presented, showing sequence similarity to members of glycoside hydrolase family 107. Using carbohydrate polyacrylamide gel electrophoresis and nuclear magnetic resonance analyses, it is shown that the fucoidanase Mef1 catalyzes the cleavage of α-linkages between fucose residues sulfated on C2 in the structure [-3)-α-l-Fucp2S--α-l-Fucp2S- on F. evanescens fucoidan was 0.1 × 10−3 Uf µM−1. By crystal structure determination of Mef1 at 1.8 Å resolution, a single-domain organization comprising a (β/α)8-barrel domain was determined. The active site was in an extended, positively charged groove that is likely to be designed to accommodate the binding of the negatively charged, sulfated fucoidan substrate. The active site of Mef1 comprises the amino acids His270 and Asp187, providing acid/base and nucleophile groups, respectively, for the hydrolysis of glycosidic bonds in the fucoidan backbone. Electron densities were identified for two possible Ca2+ ions in the enzyme, one of which is partially exposed to the active-site groove, while the other is very tightly coordinated. A water wire was discovered leading from the exterior of the Mef1 enzyme into the active site, passing the tightly coordinated Ca2+ site.Fucoidanases (EC 3.2.1.–) catalyze the hydrolysis of glycosidic bonds between fucose residues in fucoidans. Fucoidans are a compositionally and structurally diverse class of fucose-containing sulfated polysaccharides that are primarily found in brown seaweeds. Here, the structural characterization of a novel endo-α-fucoidanase, Mef1, from the marine bacterium  Enzymatic hydrolysis will therefore result in the release of a population of homogeneous fucoidan oligosaccharides, the chemical structures of which are dependent on the specific fucoidanase and fucoidan substrate.Fucoidanases, including endo-α-fucoidanase  belong to glycoside hydrolase family 107 (GH107) in the CAZy categorization and the endo-α-l-fucoidanases (EC 3.2.1.211) belong to GH168, with some categorized into the recently established GH174 -Mariniflexile fucanivorans -fucoidanase -α-l-Fucp2S--α-l-Fucp2S--fucoidanase was MfFcnA from the marine bacterium Psychromonas P5AFcnA are the only fucoidanases that have been structurally determined at high resolution , with Ca1 and Ca2 in the catalytic D1 domain and the other three (Ca3–Ca5) in the IgR domains  and Fourier transform infrared spectroscopy (FTIR).In the present study, we describe the reaction selectivity and the detailed structure of the novel GH107 endo-fucoidanase Mef1 from the marine bacterium 2.2.1.F. vesiculosus was purchased from Sigma–Aldrich, Steinheim, Germany. Fucoidans from F. evanescens  and S. latissima  were extracted using an enzyme-assisted method and fractionated by ion-exchange chromatography using DEAE resin as described previously  were extracted by a chemical method as described previously  connected to an RI-101 refractive-index (RI) detector . Samples were separated on a Shodex SB-806 HQ GPC column (300 × 8 mm) equipped with a Shodex SB-G guard column (50 × 6 mm) with elution using 100 m2.3.BLASTp analysis against a set of GH107 endo-fucoidanases. The N-terminal signal peptide was predicted using the SignalP 5.0 server . Protein sequence comparisons were performed using Clustal Omega  for global alignments. The endo-fucoidanases P5AfcnA , P19DfcnA , FWf3 , Fp273 , FFA2 , Fhf2 , Fhf1 , FWf4 , MfFcnA , FFA1 , FWf2 , Fp279 , FWf1 , Fp277 , SVI_0379 , Fda1 , Fda2  and Mef2  were used for sequence comparisons.The putative fucoidanase Mef1  was identified using MAFFT and visualized using Jalview . ESPript 3.0  for comparisons.Conserved residues of the catalytic domain were identified by multiple sequence alignment using Muscle v.3.7 with default settings, with the removal of gaps. The curated sequences were used to build a maximum-likelihood phylogenetic tree using phyML with default settings. The phylogenetic tree was statistically supported by approximate likelihood-ratio testing using default settings and values between 0 and 1 were obtained together with bootstrap values using https://www.phylogeny.fr. FigTree was used for visualization and fine adjustments of the tree. Protein domains were predicted using InterProscan (http://www.ebi.ac.uk/interpro/search/sequence/)For the phylogenetic analysis, the sequences were aligned using 2.4.Escherichia coli expression and subcloned into the pET-28b(+) vector between the NheI and XhoI restriction sites by GenScript, Piscataway, New Jersey, USA. The construct (GenBank OQ831695) was transformed into E. coli strain DH5α  for plasmid propagation using LB (lysogeny broth) agar plates with kanamycin and incubation at 37°C overnight.The Mef1 gene construct was designed without the N-terminal signal peptide but with a C-terminal 10×His tag. The Mef1 fucoidanase was codon-optimized for 2.5.E. coli BL21 (DE3) cells harboring the Pch2 (pGro7) plasmid . For expression, LB medium with 50 µg ml−1 kanamycin, 34 µg ml−1 chloramphenicol and 0.05%(w/v) arabinose was inoculated with an overnight culture in the same medium and incubated at 37°C and 180 rev min−1. Once the culture reached an OD600 of 0.6–0.8, it was cooled to 20°C and incubated at this temperature for 2 h at 180 rev min−1 before adding 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), after which the culture was incubated overnight at 20°C and 180 rev min−1. The cells were collected by centrifugation  and the cell pellets were stored at −80°C. After thawing, the cells were resuspended in 20 mM Tris–HCl buffer, 250 mM NaCl, 20 mM imidazole pH 7.4. The cells were then lysed on ice by three cycles of sonication  in the presence of 0.2 mg ml−1 lysozyme . After centrifugation  the supernatant was filtered (0.22 µm) and mixed with Ni2+–NTA Sepharose resin  pre-equilibrated with equilibration buffer . To maximize binding, the resin suspension was incubated at 4°C overnight with gentle agitation. The resin was then washed three times with buffer  and packed onto a column. The recombinant protein was eluted with 20 mM Tris–HCl buffer, 250 mM NaCl pH 7.4 and increasing concentrations of imidazole (100–500 mM). Fractions containing the pure enzyme were pooled and desalted using PD-10 columns  equilibrated with 20 mM Tris–HCl buffer, 100 mM NaCl pH 8.0 to remove the imidazole. Protein concentration was measured by the Bradford assay using bovine serum albumin as a standard , proteins were incubated at 95°C for ∼10 min. The Precision Plus Protein Standards (Unstained) molecular-weight marker (Bio-Rad) with molecular weights of 10–250 kDa was used as a standard ladder. Gels were stained using Coomassie Brilliant Blue (Bio-Rad). After separation by SDS–PAGE, proteins were transferred onto a PVDF blotting membrane  which had been pre-wetted with ethanol for a few seconds, and blotting was then performed in Tris–glycine running buffer pH 8.3 at 100 V for 45 min on ice. After protein transfer, the membrane was blocked with 2% skimmed milk in 0.01 M Tris base sodium chloride pH 7.6 (TBS) buffer for 60 min and then incubated with monoclonal anti-polyhistidine peroxidase-conjugated antibody  for another 60 min. The membrane was washed in TBS buffer three times for 20 min, and the Western blot was developed by staining with the AEC Kit following the manufacturer’s protocol (Sigma–Aldrich). Precision Plus Protein Dual Color Standard (Bio-Rad) was used as the molecular-weight marker.The purity and molecular weight of the recombinantly expressed proteins were assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) using 12% acrylamide gels according to the Laemmli protocol  glycerol and 0.02%(w/v) phenol red in a 1:1 ratio] was added to all samples before loading onto the C-PAGE gel. C-PAGE was run on a 20%(w/v) acrylamide/bisacrylamide electrophoresis gel with 100 mM Tris–borate buffer pH 8.3 for 90 min at 30 mA. Gel staining was performed in a solution consisting of 0.05% Alcian blue 8GX  in 2% acetic acid and 0.01% O-toluidine blue (Sigma–Aldrich).The fucoidanase activity was monitored using carbohydrate polyacrylamide gel electrophoresis (C-PAGE) analysis. The reaction consisted of 0.12 mg ml2.8.w/v) fucoidan from F. evanescens in 20 mM Tris–HCl buffer pH 8.0, 100 mM NaCl, 10 mM CaCl2 at 40°C according to Tran, Perna et al.  on fucoidanase activity was studied by changing the assay temperature. Likewise, the influence of NaCl concentration (25–500 mM) on fucoidanase activity was investigated at different levels of NaCl addition to the assay.Substrate-specificity screening experiments were performed using different fucoidan substrates at 0.9 mg mlM buffer concentration. The influence of pH (4–10) on fucoidanase activity was examined using buffers with different pH values .The influence of different buffers  at pH 8.0 was investigated at 0.02 2+, Mg2+, Mn2+, Cu2+, Fe2+, Zn2+, Co2+ and Ni2+) on activity was investigated by the addition of 10 mM of each cation after treatment with EDTA (2 mM) and EDTA removal by treatment with PD10. Time-course experiments were performed using 0.12 mg ml−1 Mef1 in 0.02 M Tris–HCl buffer pH 8.0, 50 mM NaCl, 10 mM CaCl2 and 0.9 mg ml−1 fucoidan from F. evanescens. The reactions were incubated for 48 h. All assays were performed for 2 h unless stated otherwise. The results were visualized by C-PAGE.The influence of divalent cations (Ca2.10.M) was determined by measuring the absorbance at 280 nm using a calculated molar extinction coefficient computed using ProtParam from ExPASy. Samples were added to 10 µl capillary tubes and analyzed using a Prometheus NT.Plex nanoDSF instrument . Thermal stability was monitored using a temperature gradient of 25–80°C with an increase of 1°C min−1 to obtain denaturation profiles. Raw data were exported into data sets that contained fluorescence at 330 and 350 nm (F330 and F350) as well as the ratio of these values (F330/F350) and absorbance at 350 nm (A350). The first derivatives of F330/F350 were plotted to visualize denaturation. The peak of the first derivative gives the value of the melting temperature (Tm), at which half of the protein is unfolded.Differential scanning fluorimetry (DSF) was carried out to determine the thermal stability of the Mef1 protein. The concentration of Mef1  to precipitate the medium-molecular-weight fucoidan products (MMP) and in this way separate them from the low-molecular-weight fucoidans (LMP), followed by centrifugation at 19 000g for 20 min. The supernatant containing LMP was evaporated under an air flow. Both MMP and LMP were solubilized in distilled water and lyophilized. The products were analyzed by C-PAGE and NMR spectroscopy; the yields and monosaccharide compositions of MMP and LMP are shown in Supplementary Table S3 , were dissolved in 500 µl 2H2O. All NMR spectra were collected using an 800 MHz Bruker Avance III instrument equipped with a TCI cryoprobe (5 mm) by acquiring 1H–1H TOCSY , 1H–1H COSY , 1H–13C HMBC (2048 × 128 complex data points sampling 256 and 6.3 ms) and 1H–13C HSQC (2048 × 512 complex data points sampling 160 and 21.2 ms).Fucoidans from F. evanescens fucoidan by Mef1 was followed using 0.005 mg ml−1 enzyme and 0.9% substrate in 10 mM Tris–HCl buffer pH 8. A time series of 1H–13C HSQC spectra  was acquired at 25°C with a non-uniform sampling of 30% of the data points in the indirect dimension. All NMR spectra were processed with ample zero filling and baseline corrections in all dimensions using Bruker TopSpin 3.5 pl7 and were analyzed using the same software.The time-dependent degradation of 2.13.−1) with elution in 20 mM degassed Tris–HCl pH 8.0 containing 50 mM NaCl on a chromatography system using a high-resolution Superdex 200 10/300 GL column and a flow rate of 0.75 ml min−1. Mef1 was detected by UV absorbance at 280 nm.Analytical size-exclusion chromatography (SEC) was used to confirm the molecular size of the protein and the state of oligomerization. SEC was performed using 200 µl enzyme . Crystallization trials of Mef1 were performed at 18°C using the vapor-diffusion method in sitting drops consisting of a 1:1 ratio of pure protein  and precipitant solution  in the presence or absence of divalent ions (1 mM Ca2+ or 1 mM Cu2+). After obtaining initial Mef1 crystals using 18% PEG 3350, 0.1 M 3-(cyclohexylamino)-2-hydroxy-1-propane sulfonic acid (CAPSO) pH 9, 4.8% 2-propanol, 17% PEG 400, the conditions for crystallization were optimized using grid optimization screens in 24-well sitting drops at 18°C using the hanging-drop vapor-diffusion method. The optimized parameters included screening of the 2-(cyclohexylamino)ethanesulfonic acid (CHES) and CAPSO concentrations, different concentrations of the enzyme  and three different dilutions  of protein and crystallization solution .Purified Mef1 protein after SEC was concentrated to 16 mg ml−1 Mef1, 1 mM CaCl2, 50 mM NaCl, 20 mM Tris pH 8.0 in a drop consisting of 24% PEG 3350, 0.1 M CAPSO pH 9, 3% 2-propanol, 4% PEG 400. The crystals were mounted and soaked in 18% ethylene glycerol and flash-cooled to cryotemperatures using liquid nitrogen.Optimal crystals of Mef1 were obtained using 12 mg mlP63 at 1.8 Å resolution. The structure of Mef1 was determined by molecular replacement with Phaser-MR . Data were collected in space group CB-Dock  was accomplished using PyMOL .All molecular graphics were prepared using 3.3.1.M. eckloniae strain DOKDO 007T belongs to the Flavobacteriaceae family; M. eckloniae was originally isolated from the rhizosphere of the brown alga Ecklonia kuromea and sequenced  using selected and verified GH107 fucoidanases. Mef1 was found to be a putative GH107 fucoidanase of 399 amino acids in length including a 19-amino-acid predicted N-terminal signal peptide as indicated by SignalP 6.0 .The marine bacterium et al., 2018a). D1 of Mef1 shows a broad sequence-identity span ranging from 17% to 51% when compared with other characterized GH107 fucoidanases (Supplementary Table S4). P5AFcnA (51%) and P19DFcnA (49%) had the highest sequence identity, followed by Mef2 (30%), Fda2 (22.9%) and Fda1 (21.5%) .The catalytic domain (D1) was predicted in Mef1 based on sequence alignment with MfFcnA (Vickers b).Sequence alignment  and phylogenetic analysis based on the D1 catalytic domain sequences revealed Mef1 to be grouped with Mef2, P5AFcnA and P19AFcnA Fig. 2b.Due to the limited number of deposited sequences of family GH107 endo-fucoidanases, the phylogenetic analysis was only used to provide sequence-alignment similarity estimations and does not allow firm conclusions to be drawn regarding possible subfamily clusters of the enzymes.et al., 2018et al., 2018The catalytic site residues of GH107 fucoidanases have been predicted to include a conserved histidine (His276 in P5AFcnA) and an aspartate (Asp201 in P5AFcnA) Table 2 that actThe fourth amino acid in the −1 subsite was however not conserved in Mef1, as Asn145 in P5AFcnA was an alanine (Ala130) in Mef1 Table 3. Neither3.2.SDS–PAGE of the purified Mef1 showed a band with the expected molecular weight of 45 kDa; the presence of Mef1 was verified by Western blot analysis using anti-His antibodies . The initial affinity purification yielded Mef1 with greater than 95% purity; this was followed by size-exclusion chromatography and the data corroborated that the native Mef1 protein was a monodisperse monomer in solution with an expected size of approximately 42 kDa, resulting in a single protein band on SDS–PAGE with a purity above 99% .M CAPSO pH 9, 3% 2-propanol, 4% PEG 400. The crystals formed after ∼3 days and were subsequently picked, cooled and subjected to X-ray diffraction studies to determine the structure. Attempts to co-crystallize the crystals with fucoidan substrate and/or products were unsuccessful.Optimal crystals formed in hanging drops with 24% PEG 3350, 0.1 3.3.8-barrel structure with a similar structural architecture to that observed for the homologous D1 domain of the GH107 endo-fucoidanases P5AFcnA (PDB entry 6m8n) and MfFcnA (PDB entry 6dlh) . The active site of Mef1 was situated at the center of the β-barrel and included the catalytic amino acids His270 and Asp187 and the three conserved amino acids of the −1 subsite Tyr128, Asn215 and Trp318 ; numbering of the sugar-binding subsites was performed according to the classical nomenclature originally proposed for endo-acting glycoside hydrolases . The active site is located in the center of an elongated groove presented on the surface of Mef1. The amino acids in the groove are largely positively charged, as is especially evident for Arg35, and are likely to assist with the binding and positioning of the largely negatively charged sulfated fucoidan polysaccharide. Indeed, Arg35 appears to be involved in the binding of the sulfate group of CAPSO by Mef1 .A proline conserved in GH107, Pro313 in Mef1, was found near the active site, where it forms a CH–π interaction with Trp318 and Pro313 in the −1 subsite and is therefore likely to be important for the proper positioning of Trp318 in the −1 subsite Fig. 3a. The a3.3.1.2+ ions were identified in the crystal structure of Mef1  was found in a very well organized Ca2+ site coordinated by Asp242, Asp246 and Asp248 . The Ca1 site is further stabilized by hydrogen bonds from Tyr252 to Asp242 and from Ser282 to Tyr252 . The Ca1 site thus seems to be very tightly coordinated; interestingly, the backbone of Arg250 is likely to be involved in bifurcated hydrogen bonding to two ligand water molecules, which coordinate Ca1, while the carboxyl group coordinates via hydrogen bonds to Asp248. A possible additional Ca2+-binding site (Ca2) was found close to the active-site residue Asp187 .Densities corresponding to two Caf1 Fig. 3: a Ca2+ 8 Table 4 and by ads Fig. 3c. The C52 Fig. 3c. The C87 Fig. 3d.d and Table 4f and 3g). CAPSO, which is normally used as a buffer, is a small, sulfated molecule and the sulfate group of CAPSO 1 is coordinated in the Mef1 crystal primarily through Arg35, while the EG site is close to a small α-helical structure stabilized by Ca1. The latter is likely to act as a dipolar charge combined with coordination from both active-site amino acids His270 and Trp318 to stabilize the EG.The coordination of Ca2 was mediated by the backbone carbonyl groups of Ala136, Ser138 and Thr140 and the amide group of Asn142  Arg53 is in a different position further away from the lining of the central active-site β-barrel than Arg35 in Mef1. Although we did not find conservation in the sequence alignment in other GH107 fucoidanases, structural conservation is possible with arginine present in this other position. Indeed, there is sequence conservation in this position in some of the other investigated GH107 fucoidanases in an alignment of P5AFcnA, P19AFcnA, Fda1, Fda2 and Svi_0379 . While the primary structures of Mef1 and P5AFcnA (PDB entry 6m8n) share a sequence identity of only 53%, the crystal structures share strong structural similarity, with an overall r.m.s.d. of 0.7 Å when superimposed. The major deviations between the two crystal structures were mainly found in the loop regions towards the putative +1 subsite. The deviations identified are numbered #1–#5 and are visualized in Fig. 3b). Deviation #1 from residues Ser135 to Asp145 forms a coil that coordinates a Ca2+ site in Mef1, while in P5AFcnA this region forms a short α-helical structure that is unable to coordinate any divalent metal ions. The second deviation (#2) from Asn215 to Tyr228 in Mef1 is close to the active site and forms a coiled structure in both Mef1 and P5AFcnA; however, it is extended in Mef1.The possible sulfate site, coordinated by Arg35 (CAPSO 1), is not conserved in the sequence alignment with P5AFcnA. In the sequence alignment, Arg53 was present two residues away from the equivalent site in Mef1 . However, in the P5AFcnA structure  extending from Ser256 to Tyr263. P5AFcnA presents an additional two-sheet β-sheet structure at the fourth deviation, while residues Asp295–Lys308 in Mef1 form a shortened random-coil structure in Mef1. The site of the fifth deviation (#5) indicates the presence of a Ca2+ sites revealed poor conservation compared with other described GH107 members, the Ca1 site is almost conserved in P19AFcnA except for Asp240 being an alanine and being completely conserved in P5AFcnA (Table 46m8n) it was evident that density was present in this location, which could be a sodium molecule. The crystallization conditions for P5AFcnA did not contain Ca2+  was however refined as an alanine in the crystal structure, although written correctly as an aspartic acid. When properly refined, we found structural conservation in the Ca1 coordination in P5AFcnA and Mef1.While the alignments of the two Mef1 CaA Table 4. From in2+ site previously predicted in the P5AFcnA crystal structure was described as weakly bound by an N atom of Lys259 and a water molecule . This water wire is lined by a hydrophilic and a hydrophobic stretch of amino-acid residues stretching from Ca1 to the EG molecule, which is in contact with the active-site residues Trp318 and His270 . This water wire might be involved in carrying the excess proton from the hydrolytic catalysis of fucoidan.A water wire was identified leading from the exterior of the Mef1 protein, closely passing the free coordination site of Ca1 and further passing directly into the active site  and formed at different times; with CAPSO the crystals formed after 3–4 days, while with CHES they formed later after around 7–10 days. Investigating the effect of these two buffers on Mef1 enzyme activity resulted in inhibition of the Mef1 enzyme. After 24 h incubation with CHES, the enzyme was inactivated at 250 mM concentration, while the enzyme was inactivated after 30 min of incubation at 250 mM CAPSO.The possible CAPSO molecule in the active site of Mef1 could be important for the crystallization of Mef1, since Mef1 crystals could only be obtained when adding CAPSO, or a small analog called CHES, to the crystallization mixture. CAPSO and CHES were investigated for possible inhibitory effects on the fucoidanase activity of Mef1 by C-PAGE analysis. Interestingly, increasing concentrations of both molecules showed an inhibitory effect on Mef1 activity . At concentrations of over 250 and 600 m3.4.F. evanescens containing alternating α- and α-glycoside bonds was efficiently depolymerized by the Mef1 enzyme -glycosidic bonds - and α-linked fucosyl residues which are differentially sulfated at C2 and/or at C4, as well as α- and α-linked galactosyls . According to the HP-SEC/RI analysis, the average molar mass of fucoidan-derived products decreased over time from approximately 650 to 8 kDa, corroborating that Mef1 is an endo-acting enzyme .A time-course experiment of the hydrolysis of fucoidan from 3.4.1.F. evanescens fucoidan were verified in a real-time in situ tracking assay using 1H–13C NMR spectroscopy . A lack of change in the signals indicated that the structural motifs were far from the cleavage site.The products of the action of Mef1 on py Fig. 5. The anoon Fig. 5a. A lacF. evanescens fucoidan, the emergence of reducing-end signals with single sulfation at the C2 position and glycosidic linkages to the C3 position was paralleled by changes to structural motifs containing 3)-α-Fucp-2-SO3−--α-Fuc-2-SO3−-, indicating that the α-glycosidic bonds in this motif, which was also partly acetylated, were hydrolyzed. In contrast, structural motifs containing 3)-α-Fucp-2,4-di-SO3−--α-Fuc-2-SO3−- repeating units remained intact .In Mef1 cleavage of ct Fig. 5a.F. evanescens fucoidan after Mef1 hydrolysis. The low-molecular-weight hydrolysis products (LMP) were separated from the medium-molecular-weight products (MMP) by precipitation with 75% aqueous ethanol and were analyzed by recording a suite of NMR spectra including 1H–1H COSY, 1H–1H TOCSY, 1H–13C HSQC, 1H–13C HMBC and 1H–1H ROESY  and the H2 and H4 protons of the sulfate groups, as well as fucose methyl groups . Mef1-derived fragments of F. evanescens fucoidan were analyzed by 2D NMR spectroscopy. The signals within each spin system were assigned primarily based upon 1H–1H COSY, 1H–1H TOCSY, 1H–13C HMBC and 1H–13C HSQC correlations.The 1H–13C HMBC experiments to detect 3JCH correlations across the glycosidic bond, and were aided by comparison to ample literature data relating to NMR data of fucoidan fractions  was found to be monosulfated at C2, as shown by a characteristic H2 chemical shift, and linked to the next fucose unit at the C3 position: the −2 position at the cleavage site. This second fucose unit was predominantly acetylated at the C3 position and was sulfated at the C2 position. In contrast, the nonreducing-end unit was primarily non-acetylated . The Mef1 enzyme thus seems to selectively catalyze the cleavage of α-glycosidic linkages between non-acetylated and monosulfated units in structural motifs that contain mono-acetylated units at the −2 position. Sulfation at C2 was observed at each fucose unit from the chemical shift values for H2 of the order of 4.5–4.7 p.p.m., whereas desulfated residues would exhibit chemical shift values in the range 3.8–4.0 p.p.m. for H2. Similarly, acetylation at C3 of residue C in the tetrasaccharide product was observed from the characteristically high 1H chemical shift at the H3 position of 5.39 p.p.m.. The molecular structure of the tetrasaccharide (including an acetyl group) constituting the principal LMP upon the degradation of F. evanescens fucoidan with Mef1 was thus identified as a C2-sulfated fucose tetrasaccharide with the cleavage site of fucose −2 acetylated on C3 . The units have the same identifiers as in Supplementary Table S3.Linkage analyses of the oligosaccharide were performed using ed Fig. 5c. The MC3 Fig. 5c. The u1H chemical shift near 4.95 p.p.m.), which is strong in the intact fucoidan and in the MMP containing -3)-α-Fucp-2,4-di-SO3−--α-Fuc-2-SO3−-. The spectra showed that the MMP fraction contains few acetylated units compared with the substrate, and the MMP is apparently predominantly comprised of -3)-α-Fucp-2,4-di-SO3−--α-Fuc-2-SO3−-. Previous results have shown similar MMP products to be formed by Fhf1 on deacetylated fucoidan from F. evanescens -α-l-Fucp2S-.Overall, Mef1 is thus suggested to specifically catalyze the cleavage of α-linkages between fucosyl residues sulfated on C2 in the structural motif -3)-α-3.5.F. evanescens fucoidan product into the active site of Mef1 displayed how well the active site accommodates the fucoidan ligand . Indeed, the docking data also showed complete congruence between the positioning of one of the fucoidan sulfate groups and one of the sulfonates of a CAPSO molecule , which is in full agreement with the crystallization data showing the CAPSO 1 and ethylene glycol positions . Thus, the positioning of the sulfate groups of the fucoidan substrate is likely to contribute to the substrate selectivity and the formation of the reactive Mef1 enzyme–substrate complex. The amino acids lining the active-site cleft, including Arg35, are likely to support the correct substrate positioning. During catalysis, the sulfated fucoidan is thus expected to approach the active-site residues to allow nucleophilic attack and the catalytic cleavage of an α bond via His270 and Asp187 .Molecular docking of a hexameric C2-sulfated fucoidan containing the tetrameric nd Fig. 6a. Indeens Fig. 3h. Thus,87 Fig. 6c.3.6.a). The optimal pH of Mef1 was around pH 8.0, where all LMPs were released in clearly visible amounts compared with the other pH values . In addition, the effect of different buffers was investigated, including Tris–HCl, borate, sodium phosphate and UB4 buffer at pH 8.0. These results confirmed that both Tris–HCl and UB4 buffers are suitable for Mef1 enzyme-activity assays .Mef1 was found to be active in a wide pH range spanning pH 6.0 to 10.0 . Within the pH range 7.0–9.0 all LMPs were visible after 2 h of reaction time . The enzyme was subjected to EDTA treatment and desalting prior to the investigation of the influence of different divalent cations . Treatment with EDTA reduced the enzymatic activity as assessed by C-PAGE . It was not possible to restore the activity of Mef1 by addition of the divalent cations Cu2+, Fe2+, Zn2+, Co2+and Ni2+, while the activity was restored by the addition of Mg2+, Mn2+ and notably of Ca2+. Together with the Ca2+ sites found in the structural analysis of Mef1, this result validated that Mef1 is a Ca2+-dependent enzyme, equivalent to previously characterized GH107 fucoidanases , with the highest production of visible smaller fucoidan oligosaccharides at 37°C . As expected, the enzyme activity decreased at 40–45°C, and temperatures above 50°C inactivated the enzyme. These findings were supported by thermal stability assessment  and determination of the melting temperature (Tm) of Mef1, which was ∼43°C. When the divalent cations were removed by EDTA, the Tm was reduced to 40°C, indicating that divalent ions (likely Ca2+) have a stabilizing effect on the Mef1 structure; Tm increased further to 46°C in the presence of Ca2+ when the substrate was also present, corroborating stabilizing effects of both Ca2+ and substrate (Supplementary Table S5).The optimal temperature range for Mef1 was estimated to be between 20 and 37°C . Although the production of oligosaccharides was considerably lower on S. oligocystum fucoidan than on F. evanescens fucoidan, the detection of activity on S. oligocystum fucoidan is noteworthy, since no fucoidanase to date has been found to be active on this substrate . Mef1 is thus the first enzyme demonstrated to catalyze the hydrolysis of fucoidan from S. oligocystum.The optimal conditions for the activity of Mef1 on fucoidan from 3.7.−1 and a decrease in absorption at wavenumbers of 1250–1150 and 1500 cm−1 , FFA2 (4.0 × 10−3 Uf µM−1) and Fhf1 (1.2 × 10−3 Uf µM−1), and about ten times lower than the activity of Mef2 -specific fucoidanase rather than an α-specific fucoidanase, which leads to the suggestion that phylogenetic relationships in the GH107 family have to be considered carefully when predicting the specificities of the members according to only sequence-based phylogeny while the sequence base is as small as it is at present.8-barrel structure with an active site positioned in the β→α loops  only reports a single Ca2+-binding site, while Mef1 has two Ca2+-binding sites (Ca1 and Ca2). In Mef1, Ca1 is a highly coordinated site, with Asp242 and Asp246 contributing with their side-chain carboxyl groups, while Asp248, Arg250 and Asp240 contribute with their backbone carbonyl groups. Two water molecules complete the heptavalent coordination of the Ca1 site. The residues involved are largely conserved with the equivalent sites in P19DFcnA and P5AFcnA. Inspection of the electron-density map based on the deposited structure-factor amplitudes of PDB entry 6m8n reveals that the crystal structure of P5AFcnA has a strong difference density peak located at an equivalent site to Ca1 in Mef1, which is likely to be an Na+ ion but could easily accommodate a Ca2+ ion, although this would need further investigation. The data suggest that there might be a level of conservation of this Ca2+-binding site in this subclass of fucoidanases, while in others, such as MfFcnA, FFA2, Fhf1 and Fhf2, no conservation seems to be present according to alignment. The importance of Ca2+ ions for Mef1 activity is likely to be related to the structural stability of the enzyme since the thermal stability increases when Ca2+ is present. The mechanistic role of Ca2+ coordination in GH107 fucoidanase enzymes has not previously been investigated and no importance of the Ca2+ ions has previously been suggested. Whether Ca2+ ions contribute similar stability in other GH107 fucoidanase enzymes remains to be elucidated, but the prospect is supported by the results obtained for Mef2, where the Tm increased from 38 to 44°C on the addition of Ca2+ ions  al. 2018. There aF. evanescens. However, molecular docking  into the active site of Mef1 did not show complete congruence between the subsite positioning of the fucoidan sulfate groups and the positioning of the CAPSO sulfonate derived from the CAPSO crystallization data  revealed Arg53 of P5AFcnA two residues away from Arg35 of Mef1, but the alignment could not establish the general significance of this arginine in P5AFcnA or in the other analyzed fucoidanases. Yet, the possibility that Arg35 is central for substrate recognition and substrate docking of Mef1 finds support in the early seminal work on the structure of Alteromonas fortis ι-carrageenase , all collected on a megaplasmid  clusters of genes, organized in polysaccharide-utilization loci (PULs), that encode sets of enzymes for the degradation of complex polysaccharides, including charged polysaccharides such as pectins -α-l-Fucp2S--α-l-Fuc-2S--α-l-Fuc-2S-3Ac--fucoidanase, 8bpdPDB reference: 10.1107/S2059798323008732/jc5061sup1.pdfSupplementary Tables and Figures. DOI: