Mef2, from the marine bacterium Muricauda eckloniae, was cloned, and the Mef2 protein was functionally characterized. Based on the primary sequence, Mef2 was suggested to belong to the glycosyl hydrolase family 107 (GH107) in the Carbohydrate Active enZyme database (CAZy). The Mef2 fucoidanase showed maximal activity at pH 8 and 35 °C, although it could tolerate temperatures up to 50 °C. Ca2+ was shown to increase the melting temperature from 38 to 44 °C and was furthermore required for optimal activity of Mef2. The substrate specificity of Mef2 was investigated, and Fourier transform infrared spectroscopy (FTIR) was used to determine the enzymatic activity (Units per μM enzyme: Uf/μM) of Mef2 on two structurally different fucoidans, showing an activity of 1.2 × 10−3 Uf/μM and 3.6 × 10−3 Uf/μM on fucoidans from Fucus evanescens and Saccharina latissima, respectively. Interestingly, Mef2 was identified as the first described fucoidanase active on fucoidans from S. latissima. The fucoidan oligosaccharides released by Mef2 consisted of a backbone of α-linked fucosyl residues with unique and novel α-linked fucosyl branches, not previously identified in fucoidans from S. latissima.Fucoidans are complex bioactive sulfated fucosyl-polysaccharides primarily found in brown macroalgae. Endo-fucoidanases catalyze the specific hydrolysis of α-L-fucosyl linkages in fucoidans and can be utilized to tailor-make fucoidan oligosaccharides and elucidate new structural details of fucoidans. In this study, an endo-α-fucoidanase encoding gene,  Fucoidans are present in the cell walls of brown macroalgae and constitute the most complex class of marine polysaccharides found to date. Fucoidans are mainly composed of a backbone of sulfated fucosyl residue in addition to normally minor amounts of other sugars such as galactose, xylose, glucose, mannose, rhamnose, and uronic (mostly glucuronic) acids . FucoidaS. latissima, or sugar kelp, is gaining ground as a new commercially cultivated brown macroalgae due to its ability to grow in the Northern hemisphere, notably in the North Atlantic [S. latissima are particularly complex. However, the following four different partial structures have been reported to date: sulfated fucan, galactofucan, fucoglucuronomannan, and fucoglucuronan [Atlantic . Fucoidaucuronan .Fucoidans show promising applications in disease treatment with biological activities such as antioxidants, anticancer , anti-inFlavobacteriaceae [Alteromonadales [Mariniflexile fucanivorans SW5, was characterized in 2006 [Formosa algae KM3553 [Formosa haliotis [Wenyingzhuangia fucanilytica CZ1127 [Alteromonas sp. SN-1009 [Fucoidan-degrading organisms are mainly found in marine bacteria such as eriaceae  and Alteonadales . These bonadales  families in 2006 . GH107 c in 2006 , FFA1, ae KM3553 ,20,21, Fe KM3553 , and Fhfhaliotis , and FWfa CZ1127 . To date SN-1009 . The chaMuricauda eckloniae (previously known as Flagellimonas eckloniae) from the Flavobacteriaceae family was originally isolated from the rhizosphere of the brown seaweed Ecklonia kurome [M. eckloniae. Mef2 was shown to be the first fucoidanase acting on fucoidans from S. latissima and furthermore, the first fucoidanase releasing a novel branched fucoidan oligosaccharide from S. latissima fucoidans. The mesophilic marine bacterium a kurome . In the Through BLAST analysis using known GH107 fucoidanase sequences, the putative fucoidanase Mef2 was identified (RefSeq: WP_055392200.1). Mef2 was categorized as a discoidin domain-containing protein in NCBI. The predicted Mef2 protein was 1067 amino acids long and was found to contain an 18 amino acid long N-terminal signal peptide according to SignalP . ModularDictyostelium discoideum (slime mold) has a related domain in the cell adhesion protein discoidin. The FA58C domain in Mef2 is likely the reason why Mef2 was categorized as a discoidin domain-containing protein when deposited in NCBI.As expected from the discoidin categorization, a coagulation factor F5/8 C-terminal (FA58C) domain (IPR000421 from 464–834) was predicted in Mef2. A Secretion system C-terminal sorting domain (IPR026444 from 997–1067) or Type IX Secretion System (T9SS) domain was predicted in the C-terminus of Mef2. T9SS domains are normally associated with protein sorting to the outer membrane . These dAn invasin/intimin cell-adhesion fragment (IPR008964 from 385-680) and a bacterial Ig-like group 2 domain  were also predicted in Mef2. The superfamily comprising the Ig-like domain type, including invasin/intimin cell-adhesion domains, is common in GH107 fucoidanases ,20,22,23Several domains likely involved in carbohydrate binding were furthermore predicted in Mef2, a fucolectin tachylectin-4 pentraxin-1 (FTP1) domain (IPR006585 from 475–845), a galactose-binding-like superfamily domain (IPR000421 from 474–963) and a carbohydrate-binding module CBM6 domain (IPR005084 from 842–963). None of these domain types have previously been identified in fucoidanases, and although they might be involved in carbohydrate binding in fucoidanases, their function remains to be investigated.8-barrel fold [The previously identified fucoidanase catalytic D1 domain, comprising a (β/α)rel fold , has notrel fold . Sequencrel fold . A phyloThe conserved GH107 fucoidanase motifs ‘RxxxxxDxxxxD’ and ‘DxxxGH’, including the conserved catalytic site amino acids aspartate and histidine, were both identified within the D1 domain of Mef2 ; howeverThe alignment also revealed conserved amino acids in Mef2  , previouE. coli BL21 (DE3) with the pGro7 chaperone. Expression of Mef2 at 20 °C overnight resulted in a partially degraded protein . Mef2 was co-expressed in  protein , which w protein , resultiF. evanescens that consist of alternating α- and α-linked fucose residues [Sargassum mcclurei, Sargassum polycystum, Saccharina cichorioides, and S. latissima, the latter three of which are known to contain α- linked α-L-fucose residues [S. latissima. Together, results indicate that Mef2 catalyzes hydrolysis of α glycosidic bonds in fucoidans, consistent with the phylogenetic clustering. The substrate specificity of Mef2 was investigated on fucoidans with different structures isolated from 11 different species of brown seaweeds . Mef2 efating α1,- and α(1residues . InteresF. evanescens and S. latissima by Mef2 was monitored from 0 to 48 h by Carbohydrate-Polyacrylamide Gel Electrophoresis (C-PAGE) and High Performance Size Exclusion Chromatography (HP-SEC)  . The smaF. evanescens changed from a single broad peak of approximately 350 kDa (100 to >800 kDa) to a polydisperse peak of approximately 5–200 kDa  to two fucoidan populations, one with the most dominant molecular weight at 200 kDa (70–500 kDa) and a low molecular weight population with a most dominant molecular weight of around 1 kDa (0.6–3 kDa)  d. These F. evanescens corresponds to a tetra-saccharide of - and -linked α-L-fucosyls with each fucosyl residue sulfated at C2 (DP4) [S. latissima and F. evanescens fucoidan-derived oligosaccharides migrating furthest in the gel after Mef2 catalyzed fucoidan hydrolysis did not co-migrate with the tetra-saccharide of the standard but migrated slightly slower in the gel, suggesting slightly larger and/or less charged oligosaccharides. The lowest band in C-PAGE resulting from FFA2 catalyzed hydrolysis of fucoidans from C2 (DP4) . However2+ had been removed by EDTA  showed that Mef2 was active at pH values from 6 to 8 in the UB4 buffer but not in the borate buffer, while the optimal pH was found at 7–8 a. Mef2 w by EDTA . For fur2+ was found in the catalytic D1 domain [2+, Cu2+, Fe2+, Zn2+, Co2+, and Ni2+ did not reactivate the Mef2 fucoidanase, while Mn2+ resulted in a slight re-activation, and Ca2+ showed the best re-activation of Mef2. The effect of Ca2+ on the stability of the protein (Tm) showed an increase in Tm of 6 °C in the presence of Ca2+, resulting in a Tm of 44 °C compared to 38 °C without Ca2+  specific GH107 endo-fucoidanases  on fucoidans from anescens ,32 and tF. evanescens fucoidan degradation was monitored by FTIR  and 1300–1350 cm−1 (from 0 to 1), which indicated changes in vibrations of C–O–C stretching of the glycosidic bonds and unidentified bonds, respectively. Furthermore, a decrease in wavenumbers 1200–1250 cm−1 (from 0 to −0.5) and 1400–1500 cm−1 (from 0 to −3) was observed, indicating changes in vibrations of S=O of the sulfate group and O–C–O of carboxylate group bonds, respectively. Changes in these wavenumbers were also previously observed for the fucoidanases on fucoidans from F. evanescens [R2 value of 0.97 (f/μM) of 1.2 × 10−3 Uf/μM.To determine the fucoidanase unit of Mef2,  by FTIR . The Mefns FeF4,  showed aanescens ,32. PARA of 0.97 , which lf/μM.Hence, the specific activity of Mef2 (F. evanescens) = 1.2 × 10−3 U−3 Uf/μM), MfFcnA (2 × 10−3 Uf/μM), Fhf2 (2.4 × 10−4 Uf/μM) and FFA2 (4 × 10−3 Uf/μM) on fucoidans from F. evanescens, the Mef2 enzyme presumably works slower than FFA2 and MfFcnA, to a comparable level as Fhf1, while faster than Fhf2 [Compared to the units of the previously characterized α-specific fucoidanases Fhf1   and 1300–1350 (from 0 to 2.5), and decreases in wavenumbers 1200–1250 (from 0 to −6) and 1400–1500 (from 0 to −9), indicated that the overall change in the substrate-product solution was larger for S. latissima than for F. evanescens fucoidans. PARAFAC analysis on the Mef2 FTIR data resulted in a linear curve with the equation 0.01 = 0.0129 × conc Mef2–0.0257 and an R2 value of 0.98 (f/μM) of 3.6 × 10−3 Uf/μM.Since the Mef2 was active on fucoidans from s (SlF4) . The spe of 0.98 , which lS. latissima) was 3.6 × 10−3 Uf/μM.Hence, the specific activity of Mef2  and pooled according to total carbohydrate content and C-PAGE analysis, resulting in six fractions (MF1-6) . The MF The structure of fractions MF2 to MF6 was further investigated by NMR spectroscopy. The NMR spectra resembled a mixture of 1-3 linked α-fucosyl units  and β-1,4 as well as β-1,6 linked galactosyl units predominating in the MMP with NMR spectral characteristics corresponding to structures previously described [S. latissima  and an equivalent deshielding of 1H2 in unit J . Hence, the preparation was predominantly acetylated at O3 of unit I and O2 of unit J.In the higher molecular weight fractions MF3-MF6), three separate spin systems were observed that resembled spin systems present in the substrate fraction of atissima d. Two ofatissima . The thi-MF6, thr1H-1H COSY, 1H-1H TOCSY, 1H-13C HMBC, 1H-13C H2BC, and 1H-13C HSQC correlations. As fucose is a 6-deoxygalactose, it shares the coupling pattern of galactose between hydrogens one and five, with a small coupling of approximately 1 Hz between the hydrogen atoms four and five. Spin systems were thus primarily assigned with TOCSY and heteronuclear NMR to identify CH groups for atoms 1-4 in each fucose residue. C4 could then be correlated to the CH groups five and six through HMBC correlations from the methyl group. A total of ten spin systems were identified and designated A-H, as shown in 3−)--α-L-Fucp-(2OSO3−)--α-L-Fucp--α-L-Fucp-(4OSO3−)--α-L-Fucp-(2OSO3−)--α-L-Fucp-(OSO3−) with branches α-L-Fucp-(4OSO3−)-(1→ connected to α-L-Fucp-(2OSO3−) at C4 of two of the units .The LMP were further separated by IEX to obtain pure oligosaccharides. In total, four fractions (OF1-4) were obtained, as determined by total carbohydrate analysis via the phenol-sulfuric acid method  Figure  and C-PA1H-13C-HMBC and 1H-1H NOESY spectra to detect 3JCH correlations and NOES across the glycosidic bonds, yielding the sequential assignment shown in 13C chemical shifts but less so with their 1H chemical shifts. Six of the eight residues additionally showed a characteristic deshielding of 1H positions (to approximately 4.5 ppm) and the corresponding 13C signals at these positions, strongly indicative of the attachment of sulfate groups at these secondary alcohol sites. In contrast, acetyl groups were not present, as witnessed by the absence of acetyl 1H NMR signals near 2 ppm and of attachment sites at secondary alcohol CH groups with strongly deshielded 1H. A sequential assignment of the spin systems was achieved primarily using S. latissima fucoidans [1H-13C HSQC and 1H-13C HMBC spectrum. Beyond correlating to C5 atoms with characteristic chemical shifts near 67 ppm, the methyl protons at C6 also correlate to C4 in the 1H-13C HMBC spectrums. Two characteristic C4 positions can be identified with deshielded 13C nuclei (80.85–81.46 ppm) but non-deshielded 1H, indicative of glycosidically linked C4 positions. These C4 positions accordingly show 3JCH correlations in the 1H-13C HMBC spectrum to anomeric CH groups of other spin systems -linked backbone at the C4 position, in contrast to previous findings of only C2 branchings in fragments of -linked b1H-13C spectra from another fraction (OF2) resembled the spectra for OF1 with some differences; however, a new reducing end was formed in this structure, with signals for the OF1 glycosidically linked residue E vanishing, the reducing end signal shifting due to the presence of a new reducing end, and signals for residues D and H exhibiting two sets of signals, indicative of their vicinity to the new OF2 α- and β-reducing end residue E -specificity, supported by NMR analysis as well as substrate specificity and phylogenetic clustering. Mef2 is the first characterized fucoidanase shown to be able to hydrolyze the very complex fucoidans from F. evanescens were subjected to NMR spectroscopy. The results indicated a backbone of α(1-3)- and α(1-4)-linked fucosyl units at comparable amounts, as follows: the chemical shifts in these fragments were consistent with the linear polymer fraction that was previously isolated as a minor component from fucoidans from F. evanescens with enzyme preparations from a marine mollusk [Additionally, the low molecular weight products released by Mef2 from fucoidans from  mollusk . The fuc2+ dependent. Previous findings showed that the endo-α fucoidanases Fda1 and Fda2 have a slightly lower optimal temperature of 30 and 32 °C, respectively [2+, resulting in an increase of Tm from 38 °C to 44 °C in the presence of Ca2+, consistent with the general Ca2+ dependency of known GH107 fucoidanases and the hypothesized function in stabilization of the enzymes [The Mef2 endo-fucoidanase was found to have optimal activity in the neutral or slightly alkaline pH range and an optimal temperature of about 35 °C. Mef2 had optimal activity at salt concentrations of 100–400 mM and was Caectively ,20,22,23F. evanescens and showed that the spectral changes upon increasing concentrations of enzyme resembled the results obtained from the Fhf1 fucoidanase, giving a specific activity of 1.2 × 10−3 Uf/μM and a lower activity than the endo-fucoidanases MfFcnA and FFA2, while a higher activity than Fhf2 [S. latissima for the first time and indicated that Mef2 activity results in larger spectral changes on fucoidans from S. latissima than on fucoidans from F. evanescens. These spectral changes could be related to changes in vibrations in both the substrate and products. Due to the complex structure of S. latissima fucoidans, the large spectral changes might be related to changes in the substrate/product rather than being the direct result of glycosidic cleavage. The activity of Mef2 was analyzed by FTIR on fucoidans from han Fhf2 ,32. In aS. latissima and F. evanescens, respectively, and was highly selective for fucose, not allowing galactose in the oligosaccharide products, as supported by NMR. NMR analysis showed that MMP were a mixture of galactofucans, resembling previously reported structural motifs from S. latissima fucoidans [Mef2 interestingly showed activity on both branched and unbranched fucoidans from ucoidans . Togetheucoidans . In the F. vesiculosus or U. pinnatifida, indicating that sulfate position and degree of sulfation influence Mef2 activity. In F. evanescens C2 and C4, sulfations predominate [F. vesiculosus have been found with many different sulfation patterns, including sulfates on C2, C2/C3, C2/C4 or C4 [U. pinnatifida are, moreover, assumed to be rich in 2,4-disulfate substitutions [S. latissima fucoidans are sulfated at C2 and/or at C4 and C3 [S. latissima fucoidans are C2 or C4 mono-sulfated.Further selectivity of Mef2 is supported by the substrate specificity since no activity was detected on other substrates with α1,3)-linkages, including dominate , while fC4 or C4 ,38. The itutions , while S-linkagesF. evanescens fucoidan product of an endo-α-fucoidanase from the marine mollusk Lambis sp. with the backbone α-L-Fucp-(2OSO3−)--α-L-Fucp-(2OSO3−)--α-L-Fucp-(2OSO3−)--α-L-Fucp-(2OSO3−) with an α-L-Fucp--branch [S. horneri by the FFA1 fucoidanase released branched oligosaccharides with the backbone structure α-L-Fucp--α-L-Fucp--α-L-Fucp--α-L-Fucp and the side chain of α-L-Fucp--α-L-Fucp-1→ at C4 of one main fucosyl residue [S. latissima have exclusively α-linked backbones.Some other branched fucoidan oligosaccharides that can be released by fucoidanase activity have been described previously, including the )-branch . In anot residue . Both ofS. latissima and reports new structural features for fucoidans from S. latissima. The LMP were sulfated oligosaccharides of α-linked fucose residues and branched at the new C4 position by single mono-sulfated fucose residues. In contrast, the sulfated fucan portion of fucoidans from S. latissima was previously only reported to be branched at C2 by single fucose residues [S. latissima seaweed, or simply the different geographic areas from which they grew, since fine-structure differences are common in fucoidans from different locations [S. latissima. The obtained data indicate that Mef2 can selectively hydrolyze the α--glycosidic bonds in the sulfated fucan part of fucoidans from S. latissima.The current study is the first report of fucoidanase activity on fucoidans from es of α1,-linked fOnly a few of the fucoidanases discovered to date share the exact specificity with regards to glycosidic linkages, branching, and sulfate pattern, while most fucoidanases are unique. This might be expected due to the large structural complexity of fucoidan molecules within and between species of brown seaweed. For a complete degradation of all the different linkages, with different sulfation patterns and branching in fucoidans, a whole battery of hydrolytic enzymes is hence likely necessary, and only a few of these enzymes have been characterized to date.F. vesiculosus was purchased from Sigma-Aldrich . Fucoidans from S. mcclurei, T. ornata, S. polycystum, H. cuneiformis, S. oligocystum, and S. serratum were extracted by a chemical method according to Bilan et al. (2002). Briefly, seaweeds were treated with 0.1 N HCl for 3 h at 70–85 °C to obtain water-soluble polysaccharides. The soluble extract was then treated with 2% CaCl2 to remove alginates. The fucoidans were then precipitated from the supernatant by 1% hexadecyltrimethylammonium bromide . The fucoidan-cetavlon precipitate was isolated by centrifugation and washed with water stirred with 20% ethanolic NaI solution for 2–3 days at room temperature, washed with ethanol, and dissolved in water. The solution was dialyzed, concentrated, and lyophilized [Fucoidans from the brown alga philized  and thenphilized . For allphilized , was usephilized  with sulphilized , see SecF. evanescens were extracted using an enzyme-assisted method and further fractionated according to the method previously described [S. latissima seaweeds from Ocean Rainforest were extracted by the same enzyme-assisted method with slight modifications. In short, seaweed was treated by a mixture of the commercial cellulase blend Cellic®CTec2 (5% v/w) and the alginate lyase SALy (0.5% w/w) (the latter prepared by heterologous expression in E. coli as described previously in [2 and removed by centrifugation. The fucoidans were precipitated by the addition of 96% ethanol (EtOH) in a fucoidan:EtOH (v/v) ratio of 1:3 and lyophilized. The fucoidan extracts were further separated by DEAE-Macroprep resin  column [Fucoidans from escribed , with anously in ) in 40 m) column  into fou2SO4 (5 mg/mL) at 30 °C for 1 h in water bath and then the mixture was diluted to 4% H2SO4 by adding water. The hydrolysis was continued for 40 min at 120 °C in autoclave. The hydrolysates were filtered through a 0.22 µm syringe filter and used for monosaccharide analysis [Monosaccharide compositions of the fucoidans were analyzed as described previously . Fucoidaanalysis . Chromat2 in 15% PEG6000 reagent was added. The mixture was allowed to stand for 35 min. The released BaSO4 suspension was measured at 500 nm in a microplate reader . BaSO4 was used as standard to generate a linear standard curve for the sulfate response. The sulfate content was determined by the turbidimetric method . In totaThe molecular weights (MW) of fucoidan fractions were determined by HP-SEC using an Ultimate iso-3100SD pump with WPS-3000 sampler  connected to an ERC RefractoMax 520 refractive index detector  . The fucM. eckloniae by BLAST using known GH107 fucoidanase encoding genes. The Clustal Omega service using HHalign algorithm  [The amino acid sequence of Mef2  was identified in the genome of ch 2022)  was usedhttp://www.cbs.dtu.dk/services/SignalP/, accessed on 17 June 2020) and the InterProScan V5 , respectively. Pairwise alignments of Mef2 were performed using the MAFFT algorithm  and Jalview software [The signal peptide sequence and the protein domain predictions were performed using the SignalP5.0 server  harboring the Pch2 (pGro7) plasmid . The LB broth contained 50 μg mL−1 kanamycin and 34 μg mL−1 chloramphenicol, while 0.05% (w/v) arabinose was used to induce expression of the pGro7 chaperone. The cells were cultured to reach OD600 0.6–0.8 at 37 °C and 180 rpm before enzyme expression was induced by the addition of 1 mM isopropyl-β-D-1-thiogalactoside (IPTG). After 20 hours at 20 °C or for optimal expression at 4 h induction at 37 °C and 180 rpm, the cells were harvested by centrifugation at 8000× g for 15 min and 4 °C and were re-suspended in buffer  at the ratio 1:3 (m/v). The enzyme was purified using a Ni2+ Sepharose HisTrap HP column resin  as previously described [The expression of the Mef2 fucoidanase was performed in escribed . A PD10 escribed . The enzescribed , were usC-PAGE, using small quantities of fucoidan substrate (approximately 7 mg) and enzyme, was used to determine the optimal assay conditions for Mef2, while kinetic studies were conducted using FTIR analysis, using high amounts of fucoidans .2. For full fucoidan hydrolysis, the reaction was performed at 35 °C for 24 h, while for investigating optimal reaction conditions the reactions were incubated for 2 h at 35 °C. The pH optimum determination was performed in reactions containing 20 mM buffer (UB4 buffer pH 2–8 or borate buffer pH 8–11). The temperature optimum was determined by running reactions at 20, 25, 30, 35, 37, 40, 45, 50, 55, 60, and 70 °C. Influence of divalent cations was investigated by first removing divalent cations by addition of 2 mM Ethylene DiamineteTetra acetic Acid (EDTA), desalting by PD10 followed by addition of 10 mM of different divalent cations . The influence of NaCl was investigated at different concentrations of 25, 50, 100, 150, 200, 250, 300, 350, 400, and 500 mM. Time-course experiments were performed from 0 min to 48 h. All reactions were stopped by heating at 80 °C for 10 min, and protein debris was pelleted by centrifugation .The Mef2 optimal reaction conditions were 0.12 mg/mL Mef2 in 20 mM Tris-HCl pH 8, 100 mM NaCl, 0.9% fucoidan, and 10 mM CaClv/v) solution of glycerol in water and 0.02% (w/v) phenol red. The samples (6–8 μL) were electrophoresed through a 20% (w/v) 1 mm thick resolving polyacrylamide gel in 100 mM Tris-borate buffer pH 8.3 at 30 mA for 90 min. Gel staining was performed with a solution containing 0.5% alcian blue 8 GX  in 2% acetic acid and 0.02% O-toluidine  in ethanol, for 1 h at room temperature. The gel was washed with distilled water until bands were visible.Before loading on the C-PAGE gel, the reaction was mixed with loading buffer (ratio reaction:buffer = 1:1) containing a 20% , as well as the ratio of these values (F330/F350) and absorbance at 350 nm (A350). Denaturation was visualized by plotting the first derivatives of F330/F350. The peak of the first derivative corresponds to the melting temperature (Tm), which is the transition midpoint of protein unfolding. Mef2 was treated with 2 mM EDTA followed by PD10 desalting to remove all EDTA. In total, 10 mM Ca2+ was afterward added for Tm assessments with Ca2+. The concentration of Mef2 (5 µM) was determined by measuring the absorbance at 280 nm using the calculated molar extinction coefficient computed by the ProtParam tool at ExPASy .The thermal stability of the Mef2 protein was determined using dynamic light scattering (DLS). Mef2 (5 µM) was transferred to a 10 µL capillary tube and analyzed with a nanoDSF Prometheus NT.Plex instrument . To obtain denaturation profiles, a temperature gradient of 25–80 °C with an increase of 1 °C minF. evanescens and S. latissima [F. vesiculosus, and at (0.00), 0.57, 1.14, 2.29, 3.43, and 4.57 µM for reactions on fucoidans from S. latissima.FTIR spectroscopy was used to monitor the degradation of fucoidans from atissima . Mef2 wa−1. The cuvette was kept at 42 °C and had a path length of 50 m. A 1 mL reaction mixture containing 2% weight/volume (w/v) fucoidan in 0.02 M Tris-HCl buffer pH 7.4 and 100 mM NaCl and 20 mM CaCl2. Following the addition of the enzyme, each reaction mixture was injected directly into the cuvette, and 100 spectra were acquired in a row for the reactions with F. vesiculosus and S. latissima SlF4. To export the acquired spectral data, the Foss integrator  was used. The data was then analyzed as described previously [All IR spectra were scanned using a MilkoScanTM FT2 FTIR instrument  in the range from 1000–2000 cmeviously .Parallel factor analysis (PARAFAC) was used to estimate calibration curves for all fucoidanase reactions. PARAFAC decomposition of the tensor X into three different matrices using one component. The spectral signal received from each enzyme reaction is represented by Matrix A . The number of spectra received in a continuous period of time is represented by Matrix B, the distance between spectra is 16.6 s (time mode loadings), and Matrix C represents the relationship between the change in spectra in A and the different enzyme dosages in B. (the scores of PARAFAC). In this study, FTIR-PARAFAC was used to quantify endo-fucoidanase activity, as previously described .S. latissima fucoidan fraction 3 (SlF3) was deacetylated by dissolving 1.5 g of fucoidans in 150 mL 12% ammonia solution as previously described for fucoidan from F. evanescens [2, 0.5 mg/mL Mef2 enzyme in 20 mM Tris-HCl buffer pH 8, and 100 mM NaCl at 35 °C for 24 h. The reaction was stopped by heating at 80 °C for 10 min and the precipitated enzyme was removed by centrifugation at 19,000× g for 15 min. The products were separated into two fractions, the medium molecular weight products (MMP) and the low molecular weight products (LMP) by adding cold ethanol 96% with a ratio of 3:1 (v/v) and collected by centrifugation at 15,000× g for 45 min. The ethanol in the supernatant was evaporated, thus isolating the low molecular weight products (LMP). The anescens . The yiew/v) was added to 20 µL fucoidan fraction followed by addition of 200 µL concentrated sulfuric acid. The absorbance was measured at 490 nm. Fractions containing carbohydrates were further analyzed by C-PAGE. The fractions containing the same oligosaccharides on C-PAGE were combined and ammonium bicarbonate salts were removed by evaporation and the samples were lyophilized. Further separation of the LMP was performed by applying 0.6 g/L LMP in water to a Q sepharose high-performance resin column (1 cm × 20 cm) equilibrated with water. The oligosaccharides were eluted at a flow rate 0.7 mL/min by a linear gradient of ammonium bicarbonate salt in water from 0 to 2 M. The total carbohydrate content in fractions was analyzed by the phenol-sulfuric acid method . In totaFurther separation of the MMP was performed by DEAE-Macroprep resin  column using a linear gradient of NaCl from 0.1 to 2 M. The fractions were combined based on total carbohydrate content  and C-PAS. latissima fucoidans, as well as LMP, MMP, and all the further separated oligosaccharides were analyzed by NMR spectroscopy. The samples (approximately 10 mg) were dissolved in 500 μL 2H2O, and NMR spectra were collected on an 800 MHz Bruker Avance III HD instrument equipped with a 5 mm TCI cryoprobe and a SampleJet sample changer. Oligosaccharide spectra were acquired at 25 °C. The 1H 1D NMR spectra  were acquired by summing up 16 transients. The 1H-1H TOCSY  was acquired by using a 10 kHz spin lock field during a mixing time of 80 ms. The 1H-1H COSY was acquired by sampling 2048 × 512 complex data points for 213 ms and 53 ms in the direct and indirect dimensions, respectively, while 1H-1H NOESY was acquired as 2048 × 256 complex data points by sampling the FID for 213 ms and 53 ms in the direct and indirect dimensions, respectively, and using a mixing time of 600 ms. The 1H-13C HMBC  and multiplicity-edited 1H-13C HSQC using adiabatic decoupling (2048 × 512 complex data points sampling 213 ms and 15.5 ms) were acquired alongside 1H-13C HMBC (1024 × 100 complex data points sampling 128 ms and 3 ms). Assignment spectra of the high molecular weight fractions were acquired at 50 °C with an 800 MHz Bruker Avance III instrument equipped with a 5 mm TCI cryoprobe and an Oxford magnet. All NMR spectra were processed with ample zero filling in all dimensions and baseline correction using Bruker Topspin 3.5 pl7 software. All spectra were subsequently analyzed with the same Bruker Topspin software.Native M. eckloniae, was characterized. Mef2 is the first enzyme of the GH107 family to show activity on the highly complex fucoidans of S. latissima. In S. latissima fucoidans, Mef2 specifically catalyzed hydrolysis of sulfated α-linked fucosyl residues with α-linked fucosyl branches. These fucoidan oligosaccharide structures with α branches have not previously been described for fucoidans from S. latissima, and they are the first fucoidan oligosaccharides from S. latissima released by a fucoidanase. In the present study, the novel endo-fucoidanase Mef2, from the marine bacterium


Ka values (6.9 ± 0.1 and 9.4 ± 0.1) attributable to ionization of the catalytic dyad His41 and Cys145, respectively. Our investigation of the roles of conserved active-site histidines showed that different amino acid substitutions of His163 produced inactive enzymes, indicating a key role of His163 in maintaining catalytically active SARS-CoV-2 3CLpro. By contrast, the H164A and H172A mutants retained 75% and 26% of the activity of WT, respectively. The alternative amino acid substitutions H172K and H172R did not recover the enzymatic activity, whereas H172Y restored activity to a level similar to that of the WT enzyme. The pH profiles of H164A, H172A, and H172Y were similar to those of the WT enzyme, with comparable pKa values for the catalytic dyad. Taken together, the experimental data support a general base mechanism of SARS-CoV-2 3CLpro and indicate that the neutral states of the catalytic dyad and active-site histidine residues are required for maximum enzyme activity.3-Chymotrypsin-like protease (3CLpro) is a promising drug target for coronavirus disease 2019 and related coronavirus diseases because of the essential role of this protease in processing viral polyproteins after infection. Understanding the detailed catalytic mechanism of 3CLpro is essential for designing effective inhibitors of infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Molecular dynamics studies have suggested pH-dependent conformational changes of 3CLpro, but experimental pH profiles of SARS-CoV-2 3CLpro and analyses of the conserved active-site histidine residues have not been reported. In this work, pH-dependence studies of the kinetic parameters of SARS-CoV-2 3CLpro revealed a bell-shaped pH profile with 2 p Since the advent of the 21st century, the globe has experienced three epidemics caused by coronaviruses, the most recent of which is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 first emerged among the Chinese population in the city of Wuhan in December 2019 before spreading worldwide at an exceptionally high rate. SARS-CoV-2 is responsible for coronavirus disease 2019 (COVID-19), which is characterized by influenza-like symptoms, such as fever, fatigue, diarrhea, dry cough, and shortness of breath. According to the World Health Organization, the global COVID-19 pandemic has resulted in over 600 million cases and six million reported deaths thus far . HoweverA coronavirus is a small spherical assembly with club-shaped protrusions composed of structural spike proteins that enable host cell entry . Once inPrevious studies have shown that homodimer formation is required for 3CLpro catalytic activity , 17, 18.i.e., (L/F/V)Q↓, where ↓ defines the cleavage site) (The 3CLpro active site is located at the interface between domains I and II . In contge site) . Substrage site) . Recent ge site) . Specifige site)   .Figure 1Crystallographic, biochemical, and MD studies have shown that the conformational flexibility and stability of 3CLpro are pH dependent. The potential effect of pH on SARS-CoV 3CLpro activity was first suggested by the significant differences in conformation between 3CLpro crystals grown at pH 6.0 and those grown at pH 7.6 or pH 8.0 . SARS-Cokcat) and catalytic efficiency (kcat/Km), was determined over the pH range of 5.5 to 10.0 . Fitting the data to a double-titration bell-shaped model . By contrast, H164A and H172A retained 75% and 26% of the enzymatic activity of WT 3CLpro, respectively (D). These results suggested that H163 and H172 are important for maintaining the catalytic activity of SARS-CoV-2 3CLpro. In addition to alanine substitutions, we introduced lysine, arginine, and tyrosine at H163 and H172. None of the amino acid substitutions of His163, that is, H163K, H163R, and H163Y, recovered the enzymatic activity of 3CLpro, further supporting the importance of His163 in the catalytic mechanism of 3CLpro (B). However, tyrosine substitution at His172 resulted in full recovery of enzymatic activity to WT levels (C and D).Alanine substitution at His163 (H163A) completely inactivated the enzymatic activity of 3CLpro A. By conectively D. These f 3CLpro B. HoweveT levels , C and Dkcat, respectively, whereas the kcat of H172Y was nearly identical to the WT enzyme (A). All tested mutations increased the Km of 3CLpro compared with WT, which had a Km of 67 ± 3 mM; the Km values of H164A, H172A, and H172Y were 103 ± 6 mM, 89 ± 1 mM, and 80 ± 2 mM, respectively (B). These effects indicate that the histidine residues play important roles in peptide substrate binding. Overall, the WT and H172Y enzymes had the highest and similar catalytic efficiencies (C), whereas H164A and H172A exhibited decreases in kcat/Km of 49% and 85%, respectively.Next, initial velocity studies were performed to acquire the kinetic parameters of the H164A, H172A, and H172Y mutants, which had partial or full catalytic activity compared with the WT enzyme . CompareT enzyme A. All teectively B. These ciencies C, whereaA). The melting temperature (Tm) was determined from the apex of the thermogram peak, where the WT enzyme had a Tm of 52.9 ± 0.1 °C, consistent with previously reported values . The calorimetric enthalpy  values determined from the area under the thermographic peak were similar for all variants: 185 ± 3 kJ/mol for WT, 194 ± 17 kJ/mol for H163A, 186 ± 8 kJ/mol for H164A, 209 ± 10 kJ/mol for H172A, and 187 ± 16 kJ/mol for H172Y .The effects of the histidine mutations  on the thermodynamic stability of 3CLpro were examined using differential scanning calorimetry (DSC). DSC thermograms of each enzyme were acquired in 20 mM Hepes (pH 7.4), 100 mM NaCl, and 0.5 mM TCEP, and the temperature was ramped from 15 to 75 °C at a scan rate of 1 °C/min to acquire the thermal unfolding transitions . The WT Fig. 5d values , 31. Thekcat pH profiles exhibited a half-bell shape with one ionizable residue with a pKa value of 6.8 ± 0.1, 6.2 ± 0.1, or 6.1 ± 0.2 for H164A, H172A, and H172Y, respectively . The kcat/Km pH profiles exhibited a bell shape with 2 ionizable groups with pKa values similar to those of the WT enzyme (6.9 ± 0.1 and 9.4 ± 0.2): H164A (7.1 ± 0.1 and 9.4 ± 0.2), H172A (6.5 ± 0.1 and 9.6 ± 0.2), and H172Y (6.4 ± 0.1 and 9.4 ± 0.3) . For H172A and H172Y, the first pKa was slightly lower than that of WT. Importantly, the overall kinetic parameters of H172A were lower than those of WT at all tested pH values.Given its complete loss of enzymatic activity, the pH profiles of H163 mutants of 3CLpro could not be determined. The pH profiles of the catalytically active histidine mutants H164A, H172A, and H172Y were similar to those of the WT enzyme . The kcaThe cysteine protease 3CLpro is highly conserved in all coronaviruses because of its essential role in processing viral polyproteins , 9, 10. In the present study, we identified key residues in the active site of SARS-CoV-2 3CLpro that interact with the substrate, in addition to the catalytic dyad His41 and Cys145. Among these residues, His163, His164, and His172 play important roles in binding the peptide substrate and ensuring its proper orientation for catalysis. Within the peptide substrate, the Gln residue has the largest number of binding interactions with the protease. The S1 pocket of the active site of 3CLpro is mainly formed by residues Phe140, Leu141, Asn142, Glu166, His163, and His172 from one monomer and Ser1 of the other monomer . The ionkcat/Km of SARS-CoV-2 3CLpro is bell shaped with pKa values of 6.9 ± 0.1 and 9.4 ± 0.1 , with pKa values of 6.4 and 8.3 for the catalytic dyad His41 and Cys145, respectively  and maximal enzyme catalysis . However, the pH profile of kcat yielded only one titratable group, the imidazole side chain of His41, needed for optimal enzyme catalysis, with a pKa of 6.7 ± 0.1.The pH profile of .4 ± 0.1 , which aa values . The simectively , 36, 37.ectively . By contectively . Our expKa values of <5.0 and 6.6, respectively, and thus exist in their neutral states under physiological conditions  with the side-chain carbonyl oxygen of P1 Gln of the peptide substrate at a 2.4 Å is crucial for the activity of 3CLpro of SARS-CoV-2 . Hence, In addition, His163 participates in aromatic stacking with Phe140 to support the neutral state of His163 and ensure its optimal interaction with the substrate Gln , 34. TheKa of H172 was 6.6; thus, this residue is also neutral in the optimum pH range (7.5–8.5) of WT 3CLpro . Our data provide experimental proof of the reported interaction between the imidazole of His163 (at Nε2) and the highly conserved Gln residue of the peptide substrate (D).Consequently, our results provide experimental evidence of the important roles of H163 and H172 in maintaining the catalytic activity of 3CLpro. Alanine substitution of H163 resulted in complete loss of enzymatic activity, and substitution with other amino acids did not recover 3CLpro activity , A and Bubstrate . Alanineubstrate . These rubstrate , 23. We ubstrate . H172K aubstrate D.In light of these findings, a catalytic mechanism of 3CLpro can be proposed . CatalysIn summary, our experimental data and previous MD studies support a general base mechanism of SARS-CoV-2 3CLpro in which the neutral states of the catalytic dyad residues and conserved active-site histidine residues are required for catalysis. In addition, we highlight the importance of the neutral states of His163 and His172 for achieving a fully active enzyme state.Escherichia coli BL21-CodonPlus-RIL (Stratagene) for protein expression as previously described (g for 45 min at 4 °C.The recombinant genes for WT and mutant 3CLpro were introduced into pET28b(+) bacterial expression vectors by GenScript, Inc. The vectors were used to transform escribed . The inovia SDS-PAGE.The supernatant was loaded onto a ProBond nickel-chelating column (Life Technologies) previously equilibrated with binding buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 5 mM imidazole, and 3 mM β-ME at 4 °C. The column was washed with binding buffer followed by washing buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 25 mM imidazole, and 3 mM β-ME. The Hisx6-tagged 3CLpro enzyme was eluted with 20 mM Tris (pH 7.5), 150 mM NaCl, 300 mM imidazole, and 3 mM βME. Finally, the fractions containing 3CLpro were loaded onto a HiLoad 16/600 Superdex 200 size-exclusion column  on an ÄKTA pure 25 chromatography system (Cytiva). The gel filtration column was pre-equilibrated with 20 mM Hepes (pH 7.5), 100 mM NaCl, and 0.5 mM TCEP. The final protein was collected and concentrated to 55 μM as determined by the Bradford assay, and the protein purity was assessed 2  as described previously (excitation of 360 nm and λemission of 500 nm.The enzymatic activities of WT 3CLpro and the histidine mutants were assessed by an FRET-based assay using the 14-amino-acid fluorogenic peptide substrate (DABCYL)KTSAVLQ↓SGFRKME(EDANS)-NHeviously , 41, 42.eviously . The reaf0 (EDANS). Next, the correction factor (Corr%) required to correct for the decrease in the emission signal of the fluorogenic substrate in the presence of the quencher (DABCYL) was calculated , was compared with that of EDANS in the absence of peptide substrate, fo (EDANS).To determine Corr%, the emission reduction of free EDANS at a specific substrate concentration, The values of Corr% calculated at different peptide substrate concentrations were taken into consideration when measuring the cleavage rate of 3CLpro. The effect of histidine mutations on the catalytic rate of 3CLpro was determined by measuring enzymatic activity at different enzyme concentrations ranging from 0.5 to 5.0 μM and a fixed peptide substrate concentration of 60 μM. The relative activity of each mutant was obtained from the slope of the straight line for each mutant.kcat and Km for the WT enzyme and the histidines' catalytically active histidine mutants. The concentration of the peptide substrate was varied from 20 to 500 μM at a fixed enzyme concentration. Three independent experiments were performed with triplicate measurement each to obtain the cleavage rate data that were fitted to the Michaelis–Menten equation using the global fitting analysis function in the kinetics module of SigmaPlot . The kinetic parameters, kcat and Km, were obtained, and the standard error bars were calculated from triplicate measurements of each reaction using GraphPad Prism 9.0 software . The results are presented as the mean ± SD.Next, initial velocity studies were performed to determine the kinetic parameters kcat and kcat/Km as a function of pH. GraFit 5.0 software (Erithacus Software Ltd) was used to determine the pKa values. Equation kcat with a single ionizable group resulting in a half-bell curve with zero activity at low pH and an activity plateau at high pH. Equation kcat/Km with 2 ionizable groups resulting in a bell-shaped curve with zero activity at low and high pH.The pH profiles of WT 3CLpro and the catalytically active mutants H164A, H172A, and H172Y were measured using the FRET enzymatic assay by varying the concentration of the peptide substrate from 20 to 500 μM at a fixed enzyme concentration. The reaction buffers were prepared over a pH range of 5.5 to 10 using 20 mM Mes for pH 5.5 to 6.5, 20 mM Hepes for pH 7.0 to 8.0, and 20 mM Ches for pH 8.5 to 10.0. All reaction buffers contained 100 mM NaCl, 1 mM EDTA, 1 mM TCEP, and 20% (v/v) DMSO. Initial velocity studies were performed to determine the kinetic parameters k is kcat, k(limit) corresponds to the maximum limit of kcat, and pKa is the dissociation constant of the single ionizable group. In Equation k is kcat/Km, k(limit) corresponds to the maximum limit of kcat/Km, and pKa1 and pKa2 are the dissociation constants of the first and second ionizable groups. The 3 independent pH profile measurements were analyzed using GraFit 5.0 software that provided the pKa values and standard errors.In Equation p) as a function of temperature. The Tm of 3CLpro was determined from the temperature at the apex of the thermal transition, and the calorimetric enthalpy  of the transition was estimated from the area under the thermal transition curve using NanoAnalyze Software, version 3.11.0 (TA Instruments).The effects of mutations on the thermodynamic stability of 3CLpro were assessed by DSC in a Nano-DSC instrument (TA Instruments). A fixed enzyme concentration of 25 μM was used in buffer containing 20 mM Hepes (pH 7.4), 100 mM NaCl, and 0.5 mM TCEP. All samples were scanned from 15 to 75 °C at a temperature ramp rate of 1 °C/min. The buffer was used as a reference, and the protein samples were degassed for 10 min prior to the start of each analysis run. The DSC scans were acquired by ramping up the temperature twice to obtain 2 thermograms; the second scan was used as the buffer background for each sample. The lack of signal in the second ramp-up temperature scan confirmed that the melting transitions of all 3CLpro variants were irreversible. The DSC scans were normalized for protein concentration and baseline corrected by subtracting the corresponding buffer baseline. The data were then converted to plots of excess heat capacity (CThe authors declare that all data that support the findings of this study are available within this article and its accompanying files.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


PDS and BRI1 of marker genes were chosen for targeting. CasΦ2 has the function to cleave pre-crRNA. In the presence of 10 mM Mg2+ irons concentration, sgRNA3 type guided CasΦ2 to edit target gene and generate mutation, and a mutant seedling of AtBRI1 gene with an expected male sterile phenotype was obtained. In the process of tobacco transformation, the gene editing activity of CasΦ2 can be activated by 100 nM Mg2+ irons concentration, and sgRNA1 type guided CasΦ2 to edit target gene. Mutant seedlings of NtPDS gene with an expected albino were obtained. The results indicate that CasΦ2 can effectively edit target genes under the guidance of different sgRNA type in the presence of Mg2+ ions. Together, our results verify that the CRISPR/CasΦ2 system is an effective and precise tool for genome editing in plants.CRISPR/Cas system has developed a new technology to modify target genes. In this study, CasΦ2 is a newly Cas protein that we used for genome modification in Arabidopsis and tobacco.  In crop germplasm resources, rare mutations can lead to qualitative changes in traits and increase crop yield. However, these mutations are extremely hard to obtain in natural populations . The tarCas genes in Class II have been extensively studied [Cas9 gene to be edit target genes was reported in mammalian cells [Cas genes for plant genome editing have been reported. The second most widely used Cas protein in plant genome editing is Cas12 [Cas13 and Cas14 were discovered and applied to plant genome editing [The CRISPR/Cas systems include two general classes, Class I and Class II. According to their composition, they can be further divided into 6 types and 33 subtypes. In particular,  studied ,6. In 20an cells . Howeveran cells ,9,10,11.an cells . Since tis Cas12 ,14. Subs editing ,16. Thes editing .2+ irons. CasΦ2 has a wide PAM sequence . It is confirmed that the genome editing function of CasΦ2 in vivo in plants is of great significance [CasΦ2 is a new Cas protein from huge phages. CasΦ2 is 789 amino acids and contains a C-terminal RuvC domain, and is about half the size of Cas9. CasΦ2 can process pre-crRNA into mature crRNA in the presence of Mgificance . To veriAtBRI1 and NtPDS of marker genes were successfully mutated, and mutant seedlings exhibiting the expected phenotype were observed. These results showed that a CRISPR/CasΦ2 system can be developed as a novel genome editing tool in plants. This system will benefit the research of plant functional genomics and, ultimately, crop breeding.Here, we reported the application of CRISPR/CasΦ2 system in plant genome editing. CasΦ2 gene was cloned to replace the SpCas9 gene in the binary vector pHSE401, resulting in 2 × 35S: CasΦ2 in which CasΦ2 is under the control of the 2 × 35S promoter  and Arabidopsis thaliana and synthesized commercially  [CasΦ2 gene sequence included an N-terminal 3 × Flag and N- and C-terminal nuclear localization signals (NLSs) to ensure nuclear compartmentalization in plant cells, respectively. CasΦ2 gene was directly cloned downstream of the 2 × 35S promoter cassettes from cauliflower mosaic virus (CaMV) by Xba I and Sac I sites from pHSE401 vector by replacing the Cas9 gene [Bsa I sites was replaced the SpR fragment of pHSE401-CasΦ2 vector by Hind III site to produce the CRISPR/ CasΦ2 binary vectors, the fragment was amplified with the primer pairs HSBSF/R, PCR was performed using KOD-Plus-Neo , creating pCasΦ2 vector.To construct CRISPR/CasΦ2 vector, the , China) . The Casas9 gene . Using tArabidopsis Columbia, the sgRNA and promoter sequences were amplified using overlapping PCR and the sgRNA sequence was cloned under the AtU6-26 promoter [AtBSD5 for target gene. All of the primers used in this work are listed in To construct sgRNA vector, AtU6-26 promoter sequence was amplified from promoter . The sgRAtPDS and AtBRI1 gene of Arabidopsis and NtPDS gene of tobacco as the target genes. The AtPDS gene was selected two target site, one of which was target g_33 from Pausch et al. (AtPDS2 target site) [Bsa I site, 60 ng pCasΦ2 vector and 15 ng desired sgRNA amplified fragment are combined with 35 U T4 DNA ligase (NEB), 10 U Bsa I (NEB), 1.5 µL 10 mM ATP and 1.5 µL 10 × CutSmart buffer (NEB) in a 15 µL reaction volume. The solution was incubated as follows: 37 °C for 5 min, 10 °C for 10 min, 20 °C for 5 min, final step 37 °C for 5 min, 15 cycles the conditions. Ten µL solution was transformed into Top10 chemically competent cells  and transformations were sequenced.For the target site construction of the target gene, the PAM 5′-TBN-N18)-3′  was chosen as the target site. To identify the PAM of CRISPR/CasΦ2 in plants, we constructed target gene vector for Agrobacterium-mediated plant transformation as follows. We selected -3′ , sgRNA expression cassette was then inserted into the pHSE401 vector by Agrobacterium tumefaciens strain GV3101 by freeze–thaw method. Arabidopsis Col-0 wild-type plants were infected by floral dip method. The collected seeds were screened on 1/2 MS medium plates containing 50 mg/L hygromycin.The seeds of Columbia-0 background were sterilized in ethanol for 30 s and 2.4% sodium hypochlorite solution for 5 min. The seeds were then rinsed 3 times with sterile water and were sown on 1/2 MS medium plates. After stratification at 4 °C for 2 d, the plates were transferred to a growth chamber at 21 °C with 16 h light/8 h dark. The construction vectors were transformed into Nicotiana benthamiana plants were used for transformation. Plant were grown at 26 °C with 16 h light/8 h dark in the growth chamber. The target gene vectors were transformed into Agrobacterium tumefaciens strain GV3101 and used for Agrobacterium infiltration of tobacco leaves. Protocols for transfection and regeneration were the same as Baltes et al. [2 .s et al. . The leaAtBRI1 gene was amplify with two pairs of primers AtBRI1-F/R. The target AtPDS1 and AtPDS2 fragment of AtPDS gene were amplify with two pairs of primers AtPDS1-F/R and AtPDS2-F/R, respectively. The fragment of AtBSD5 gene was amplify with two pairs of primers AtBSD5-F/R. The fragment of NtPDS gene was amplify with two pairs of primers NtPDS-F/R. PCR protocol was as the following conditions: 36 cycles of 1 min at 94 °C, 30 s at 60 °C, and 1 min at 72 °C, final step 5min at 72 °C. The PCR products were then analyzed by agarose gel, and the PCR products were sequenced to identify mutations. All of the primers are listed in To identify the mutation in plants, genomic DNA was extracted from leaf tissues of transgenic seedlings using the Maxi CTAB method  and the


Herein, we report a new fluoroborophosphate, (NH4)3B11PO19F3 (ABPF), containing four different functional units: [BO3], [BO4], [BO3F] and [PO4]. ABPF exhibits a KBBF-like structure while eliminating the limitations of KBBF crystal. The unique [B5PO10F]∞ layers enhance ABPF’s performance; for example, it has a large SHG response (1.2 × KDP) and a sufficient birefringence (0.088 at 1064 nm) that enables the shortest phase-matching wavelength to reach the DUV region. Meanwhile, the introduction of strong B-O-P covalent bonds decreases the layered growth habit. These findings will enrich the structural chemistry of fluoroborophosphate and contribute to the discovery of more excellent DUV NLO crystals.Deep-ultraviolet (DUV) nonlinear optical (NLO) crystals that can extend the output range of coherent light below 200 nm are pivotal materials for solid-state lasers. To date, KBe This paper reports a new nonlinear optical crystal that has a large second harmonic generation response, a short cutoff edge and a sufficient birefringence for deep-ultraviolet phase matching. However, its layered growth habit, resulting from large interlayer spacing (6.25 Å), and the high toxicity of raw materials are not desirable   and [POxF4-x]  units with superior microscopic properties were employed in NLO materials design, which led to the discovery of the promising NLO crystals: AB4O6F , MB5O7F3 , (NH4)2PO3F, NaNH4PO3F·H2O, etc. , [BO3F], [BO2F2], [PO4] and [PO2F2], construct the backbone of fluoroborophosphates. These compounds have large band gaps in the range of 4.34–6.45 eV, indicating their feasibility for applications in the UV or DUV region. Among them, five compounds are acentric and exhibit a moderate SHG response of 0.3–1.1 times that of benchmark KDP. Their birefringence (≤0.044) is not large enough to satisfy the DUV PM condition.Traditionally, the exploration of UV NLO crystals is mainly focused on borate and phosphate systems, and typical crystals include LiBO4 (KDP) –23. Borooperties ,24. Rece and Δn) ,25. Also2O, etc. –34. Howe2O, etc. –43. As sπ-conjugated [BO3] units into the fluoroborophosphate system to enhance the birefringence, thus regulating the PM wavelength for DUV applications. A new fluoroborophosphate, (NH4)3B11PO19F3 (ABPF), with four kinds of structural units—[BO3], [BO3F], [PO4] and [BO4]—was successfully designed and synthesized. Fascinatingly, ABPF exhibits a new type of KBBF-like structure with unique [B5PO10F]∞ layers connected by shared oxygen atoms forming the final 3D framework. It inherits the excellent properties of KBBF, such as a wide transparency range, a large SHG response and a suitable birefringence to satisfy the DUV PM condition. Beyond these, ABPF has a non-layered growth habit and is chemically benign. These properties make ABPF a promising DUV NLO crystal. In addition, the contributions of multiple anionic groups to the linear and NLO properties of ABPF were confirmed by the first-principles calculations. Our results highlight the synergistic effect of multiple anionic groups on the design of DUV NLO materials and open up new possibilities for exploring DUV NLO materials in fluoroborophosphates.In this work, we attempt to introduce 4 was found in decomposition products . The constituent elements and the anion units are further confirmed by elemental analysis and infrared (IR) spectroscopy .Polycrystalline samples of ABPF were synthesized via the high-temperature solution method in a closed system, and the photograph of ABPF crystals is shown in Supplementary Fig. 1. Crystallographic data are contained in CCDC 2153289 in crystallographic information file format. The purity of the phase was checked by powder X-ray diffraction . The results of thermogravimetric analysis-differential scanning calorimetry (TG-DSC) curves and powder XRD patterns show that ABPF begins to decompose after 180°C, and BPOR3 (see Supplementary Table 2), and the basic structure is shown in Fig. 3] triangle, [BO4] and [BO3F] tetrahedra, while one crystallographically independent phosphorus atom exhibits the coordination environment of [PO4] tetrahedron. The bond lengths, bond angles and bond valences are all in the reasonable range (see Supplementary Tables 3–8). The fundamental building block (FBB) is unique [B5PO14F], which is composed of the [B3O6F] ring and three branches: [PO4] tetrahedron, [BO4] tetrahedron and [BO3] triangle , and play important roles in its excellent NLO properties [4O6F]∞ layers in the structure of ABF, the layers in ABPF are further connected by shared oxygen atoms of [BO4] and [PO4] tetrahedra, stacking along the c-direction to form a 3D framework. Also, the interlayer spacing of ABPF is 3.97 Å, less than that of KBBF (6.25 Å), and NH4+ cations are filled in the interlayer  expressions  units contribute 96% to the birefringence, confirming that the introduction of π-conjugated coplanar [BO3] units contributes greatly to the enhancement of birefringence . As a result, the excellent comprehensive performance of ABPF, i.e. wide DUV transmittance, large SHG response and sufficient birefringence, is mainly derived from the unique KBBF-like structure composed of π-conjugated [BO3] units and non-π-conjugated [BO4], [PO4], [BO3F] units, which suggests the effectiveness of the multiple-anionic-groups design strategy. Moreover, the shortest PM wavelength of ABPF is 190 nm according to the calculation results of the refractive index dispersion curves  , shows ty (REDA)  units coπ-conjugated units and non-π-conjugated units means it exhibits excellent optical properties, namely, the highest NLO coefficients, the largest birefringence and the shortest PM SHG limit among all fluoroborophosphates. Owing to a beryllium-free, no-layered growth habit, and excellent optical properties, ABPF has a promising future as DUV NLO crystal. Moreover, we propose that the introduction of strong covalent bonds between layers can enhance the interlayer interaction force while simultaneously maintaining the large optical anisotropy of layered structures. More importantly, the emergence of ABPF once again proves the advancement of the ‘fluorination strategy’ in the DUV NLO field. These findings will facilitate the discovery of more DUV NLO materials with optimal and practical performance.In conclusion, a new type of KBBF-like compound, ABPF, with four different units, has been successfully obtained, and the synergistic effect of 4PF6 , NH4HCO3  and B2O3 . All chemicals above were used without further purification. A mixture of NH4PF6 , NH4HCO3  and B2O3  was loaded into a quartz tube , and the tube was flame sealed under 10−3 Pa. The tube was heated to 400°C for 24 h, held at this temperature for 96 h, cooled to 300°C for 150 h and cooled to room temperature with a rate of 2°C h−1. Colorless crystals can be observed at the bottom of the tube, covered with a thin layer of amorphous sticky substance. After mechanical stripping, ABPF crystals can be obtained with the yield of ∼80% based on the B element.Crystals were obtained via the high-temperature solution method in a closed system. NH2 atmosphere. The sample was placed in a Pt crucible and heated from 40 to 800°C at a rate of 5°C min−1. Elemental analysis was analyzed on the single crystal surface by a field emission scanning electron microscope  equipped with an energy dispersive X-ray spectroscope . IR spectroscopy was measured by Shimadzu IR Affinity-1 Fourier transform infrared spectrometer. The transmittance measurement of a transparent crystal was measured by Shimadzu SolidSpec-3700DUV spectrophotometer under a flowing N2 atmosphere. Powder SHG intensity was measured via the Kurtz-Perry method using a Q-switched Nd: YVO4 solid-state laser  at 1064 nm and 532 nm, for visible and UV SHG, respectively. Polycrystalline samples were ground and sieved into the following particle size ranges: 38–55, 55–88, 88–105, 105–150, 150–200 and 200–250 μm. The samples were loaded into a 1-mm-thick aluminum holder with an 8-mm-diameter hole. The sieved KDP and β-BBO samples were used as references.Powder XRD data were collected using a Bruker D2 PHASER diffractometer at room temperature. The single-crystal XRD data were collected using a Bruker D8 Venture diffractometer and the crystal structure was solved using Olex2. The interference pattern of polarized light was measured using a polarizing microscope (ZEISS Axioscope 5). TG-DSC were measured on a simultaneous NETZSCH STA 449 F3 thermal analyzer instrument under a flowing Nnwac110_Supplemental_FilesClick here for additional data file.


The as-prepared products possess a specific capacitance of 605 C g−1 at a current density of 1 A g−1. The asymmetric supercapacitor (ASC) possesses an energy density of 141.3 W h kg−1 at a power density of 2700.5 W kg−1 and capacity retention of 88.1% after 10 000 cycles, indicating its promising potential for energy devices. ZnCo2O4@PPy-50 exhibited an excellent OER performance and outstanding HER performance in alkaline media. As an advanced bifunctional electrocatalyst for overall water splitting, a voltage of 1.61 V at a current density of 50 mA cm−2 outperforms the majority of noble-metal-free electrocatalysts.Heterogeneity can be used as a promising method to improve the electrochemical performance of electrode materials; thus, ZnCo 2O4@PPy samples were prepared using a facile hydrothermal route and an electrochemical deposition process.Heterogeneity can be used as a promising method to improve the electrochemical performance of electrode materials; thus, ZnCo Thus, the design and selection of ideal electrode materials can effectively solve this problem.13 In addition, electrocatalytic water splitting has been regarded as the most hopeful method to generate high-purity hydrogen.14,15 However, the efficiency of water splitting is restricted by the kinetically sluggish oxygen evolution reaction (OER).16,17 Therefore, the construction of efficient and economical electrode materials is the key to improving the device performance and fabricating highly active catalysts.Our rapidly developing economic society requires a technological evolution to construct high-efficiency energy-storage devices for future renewable power tools.2, Co3O4, and ZnCo2O4 materials, have been widely investigated.18–21 Among them, ZnCo2O4 electrodes stand out because of their high theoretical capacitance and a high number of active sites.22,23 For example, Pail et al. fabricated a rGO–ZnCo2O4 nanocomposite through a hydrothermal method, which possesses a specific capacitance of 500.8 F g−1 at a current density of 1 A g−1.24 The Kamble group synthesized a ZnCo2O4 thin film electrode, which presents a specific capacitance of 127.8 F g−1 at a current density of 1 A g−1 and 80.6% capacity retention after 3000 cycles.25 Su and coworkers researched flower-like ZnCo2O4 with a specific capacitance of 384 F g−1 at a current density of 1 A g−1.26 Choi et al. reported a ZnCo2O4 electrode, which was fabricated through simple methods. The samples can be directly utilized as electrode materials and show excellent OER performances.27 However, the single-crystalline ZnCo2O4 electrodes possess poor cycle stability. An effective strategy for improving this is compounding with conductive polymers. As a typical conjugated polymer, PPy is considered to be a very promising electrode material due to its high electrical conductivity. At the same time, reports indicate that combining PPy with metal compounds can greatly improve the specific capacitance and cycle performance as well as decrease the overpotential. This can be attributed to PPy promoting electron transport and reaction internal resistance.Owing to their rich redox active sites and large specific surface areas, various transition metal oxides, including ZnO, SnO2O4@PPy-50 nanostructures directly grown on Ni foam using a hydrothermal method and an electrochemical deposition process. The ZnCo2O4@PPy electrode materials show a capacitance of 605 C g−1 at a current density of 1 A g−1. When used as anode materials, the as-fabricated device exhibits an energy density of 141.3 W h kg−1 at a power density of 2700.5 W kg−1 and a capacity retention of 88.1% after 10 000 cycles. As electrocatalysts, the ZnCo2O4@PPy-50 samples show a low overpotential of 240 mV at 50 mA cm−2 and a low cell voltage of overall water splitting.In this work, we synthesized novel ZnCo2.2) was immersed in a 1.0 M HCl solution for 30 min. Next, the Ni foam was repeatedly cleaned with ethanol and deionized water and then dried overnight.In the experiments, Ni foam 2·6H2O, 4 mmol Co(NO3)2·6H2O, 3 mM NH4F, and 6 mM urea were dissolved in 60 mL deionized water and magnetically stirred for 30 min. Then, the pre-treated Ni foam was placed in a uniform solution and kept at 140 °C for 6 h. After cooling to room temperature, the ZnCo2O4 precursor was washed with deionized water and ethanol and dried at 60 °C overnight. Afterwards, the sample was calcinated at 350 °C for 2 h. The average mass loading of the sample was 1.9 mg cm−2.The ZnCo2.22O4 nanowire surfaces by the electrochemical deposition method. For the cathodic deposition, a solution of 1 mL Py and 3 g p-toluenesulfonic acid was used as the electrolyte. The deposition was carried out in a three-electrode system, consisting of a piece of Ni foam with ZnCo2O4 as the working electrode, a Pt foil as the counter electrode, and Ag/AgCl as the reference electrode. The deposition was carried out at a constant potential of 0.95 V for 50 s at room temperature. The final samples were washed with deionized water and dried at 60 °C. The average mass loading was 2.0 mg cm−2.The PPy nanofilm was fabricated on ZnCo2.3−1). Scanning electron microscopy (SEM) using a JSM-7001F microscope and transmission electron microscopy (TEM) using JEM-2100 PLUS were also performed. The surface area of the samples was investigated through N2 absorption and desorption isotherms.The morphology and crystal structure of the as-prepared samples were studied by X-ray powder diffraction (XRD) using a Shimadzu-7000 with Cu Kα radiation, X-ray photoelectron spectroscopy (XPS) analysis utilizing an ESCALAB 250 with an Al Kα source, and Fourier transform infrared spectroscopy . Cyclic voltammetry (CV) curves, galvanostatic charge–discharge (GCD) tests and electrochemical impedance spectroscopy (EIS) results were measured in a three-electrode system in 3.0 M KOH solutions. The as-produced samples, Pt foil, and Hg/HgO were used as the working electrode, counter electrode, and reference, respectively.2.52O4@PPy-50 samples as the cathode and active carbon (AC) as the anode. The specific capacitance (Cs) of the electrode materials were obtained using discharge times (Δt) according to the following equation:I is the current density, m represents the mass of the electrode, and ΔV denotes the potential windows.E is the energy density, C stands for the specific capacitance, P represents the power density, and V refers to the voltage window.Asymmetric supercapacitors were constructed using the ZnCo2.6Linear sweep voltammetry (LSV) polarization curves and long-term durability measurements were obtained on a CHI 660E electrochemical workstation (Shanghai Chenhua) with Ag/AgCl as the reference electrode and Pt foil as the counter electrode, and the as-prepared samples as the working electrode in 1.0 M KOH electrolyte.3.2O4 samples is obtained using a facile hydrothermal method. Subsequently, by using an electrochemical deposition method, which employs ZnCo2O4 as the precursor under different conditions, the positive electrodes can be immediately obtained.The fabrication process of the positive electrode is schematically illustrated in 2O4 and ZnCo2O4@PPy samples are shown in θ = 44.6°, 51.8°, and 76.6°). In the blue curves, the main peaks at 2θ values of 31.6°, 36.4°, 44.6°, 59.1°, and 64.5° can be indexed to the (220), (311), (400), (511), and (440) planes of the cubic spinel ZnCo2O4 phase (PDF no. 23-1390). In the XRD pattern of the composite structure, the diffraction peaks located at the same peak position may be assigned to the ZnCo2O4 phase. In the XRD pattern of the composite material, a clear peak appears at about 26°, proving that PPy successfully coated the surface of ZnCo2O4. FTIR spectroscopy was utilized to further study the distinct structures of the ZnCo2O4, PPy, and ZnCo2O4@PPy materials  is presented at a relative pressure between 0 and 1.0, which indicates the presence of the mesoporous structure. The BET surface areas of the ZnCo2O4 and ZnCo2O4@PPy-50 samples are calculated to be 45 and 69 m2 g−1, respectively. It can be found that the ZnCo2O4@PPy-50 electrodes possess a larger surface area than the ZnCo2O4 electrode materials.The XPS pattern of the ZnCo2O4 samples possess wire-like shapes. From the high magnification SEM images, the diameter of the nanowires is approximately 20 nm. In 2O4 electrode materials. After an electrochemical deposition process, the morphologies of the ZnCo2O4@PPy-20 samples are more rough and uneven than the ZnCo2O4 products. The average diameter of ZnCo2O4@PPy-50 is around 25 nm , the CV curves of the ASCs at different voltage windows were obtained and show that the ZnCoto 1.5 V . The cur−1. As shown in 2O4, ZnCo2O4@PPy-20, and ZnCo2O4@PPy-100, the LSV curves of the ZnCo2O4@PPy-50 (η50 = 240 mV) sample possess the highest current under a certain applied voltage. To further research the OER performance, Tafel slopes were obtained and are exhibited in 2O4@PPy-50 is 46.5 mV dec−1, which is smaller than that of Ni foam (217.7 mV dec−1), PPy (76.1 mV dec−1), ZnCo2O4 (58.3 mV dec−1), ZnCo2O4@PPy-20 (75.7 mV dec−1), and ZnCo2O4@PPy-100 materials (47.3 mV dec−1). The smaller value demonstrates that ZnCo2O4@PPy-50 possesses a rapid OER reaction rate.The OER performance and HER activity of the as-prepared samples were tested using a three-electrode system in a 1.0 M KOH electrolyte. Before the measurement, the electrode materials were activated 50 times through the cyclic voltammetric curves. The OER performance was studied by LSV curves at a scan rate of 1 mV s−1. The ZnCo2O4@PPy-50 material exhibits lowest overpotential (η10 = 133 mV) at a current density of 10 mA cm−2. As we can observe in −1 for ZnCo2O4@PPy-50 is much lower than 130.5 mV dec−1 for the Ni foam, 135.9 mV dec−1 for PPy, 123.3 mV dec−1 for ZnCo2O4, 125.8 mV dec−1 for ZnCo2O4@PPy-20, and 116.4 mV dec−1 for ZnCo2O4@PPy-100. A lower Tafel slope is more favorable for HER performance. In 2O4@PPy-50 shows the smallest overpotential (OER and HER).The HER activity was measured for the same device. The LSV curves  were obt2O4@PPy-50 electrode materials show outstanding stability with no significant decrease after 10 h. As shown in 2O4@PPy-50, is 1.61 V at the current density of 50 mA cm−2. In 2O4@PPy-50 sample exhibits a steady trend after 10 h.It is important for catalysts to have excellent stability for practical application. The ZnCo2O4 nanowires on Ni foam benefits from sufficient contact between the electrode and current collector. Moreover, the PPy shell can provide high electrical conductivity pathways and accelerate the electron transportation speed. Finally, the synergetic effect between ZnCo2O4 and PPy also improves the cycling performance of the electrode materials.Combined with the electrochemical reaction mechanism , the exc4.2O4@PPy-50 electrode material was successfully grown on Ni foam, which possesses a specific capacitance of 605 C g−1 at a current density of 1 A g−1. In addition, the as-prepared samples retain 93.5% capacitance after 7000 cycles. The ASC possessed an energy density of 141.3 W h kg−1 at a power density of 2700.5 W kg−1 and capacity retention of 88.1% after 10 000 cycles. ZnCo2O4@PPy-50 exhibited excellent OER performance and outstanding HER performance in alkaline media. As an advanced bifunctional electrocatalyst for overall water splitting, a voltage of 1.61 V at a current density of 50 mA cm−2 outperforms the majority of the noble-metal-free electrocatalysts.In summary, ZnCoThe authors declare no conflict of interest.RA-010-D0RA05177B-s001