Ulvan lyase from Formosa agariphila and its applicability in depolymerisation of ulvan extracted from three different Ulva species

Algal Research 36 (2018) 106–114

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Ulvan lyase from Formosa agariphila and its applicability in depolymerisation of ulvan extracted from three different Ulva species

T

Venkat Rao Konasania, Chunsheng Jinb, Niclas G. Karlssonb, Eva Albersa,

⁎

a b

Industrial Biotechnology, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden

ARTICLE INFO

ABSTRACT

Keywords: Ulvan Green macroalgae Ulva sp. Ulvan lyase CAZy PL28 Formosa agariphila

Members of green macroalgae cause green tides in eutrophicated coastal waters. These green tides pose an environmental issue and an economic burden on coastal municipalities. The biomass from these green tides has a potential to be used as feedstock in biorefinery due to the content of interesting biomacromolecules. Ulvan, an anionic water-soluble polysaccharide, is one of such components, and its depolymerisation to high-value oligosaccharides or fermentable monosaccharides would bring value to green tide biomass which is otherwise left to decompose. However, only a few ulvan depolymerising enzymes are studied to date. Ulvan lyases depolymerise ulvan, via the β-elimination mechanism, leading to release of oligosaccharides with an unsaturated 4‑deoxy‑L‑threo‑hex‑4‑enopyranosiduronic acid at the non-reducing end. In this study, we have identified the presence of two different domains, a catalytic and a non-catalytic, in a putative ulvan lyase from Formosa agariphila KMM 3901. We overexpressed, purified, and biochemically characterised the full-length ulvan lyase, which was found to be most active at a temperature of 45 °C and pH 8.5. It exhibited high specificity for ulvan and did not degrade heparan sulphate, chondroitin sulphate, alginate, pectin or xanthan. Detailed analyses of end products of the enzymatic degradation of ulvan using 1H NMR and LC-MS revealed a disaccharide with an unsaturated uronic acid (∆) linked to 3‑sulphated rhamnose (Rha3S), trisaccharide with xylose (Xyl) flanked by Rha3S (Rha3S-Xyl-Rha3S), tetrasaccharide with an unsaturated uronic acid at the non-reducing end (∆Rha3SXyl-Rha3S) and pentasaccharides (Rha3S-Xyl-Rha3S-Xyl-Rha3S and branched ∆Rha3S-Xyl-(∆)Rha3S) as the principal end products. We also found that the catalytic domain that lacks the non-catalytic carbohydrate binding module exhibited higher affinity for the soluble ulvan and efficiently depolymerised it. This study reveals the characteristics of the endolytic ulvan lyase, which is a member of the ulvan utilisation loci in Formosa, and points towards the potential ulvan depolymerisation applications in Ulva biorefinery.

1. Introduction Green macroalgae proliferate rapidly in eutrophicated waters, causing green tides or algal blooms, which are a significant environmental and an economic problem in coastal cities [1]. The utilisation of green macroalgae biomass is currently limited, and only a small proportion is used for food under the name “sea lettuce”. The main component of green algae biomass is cell wall polysaccharides, which constitute up to 54% of the total dry weight [2]. Among these, the structural polysaccharide ulvan is the most abundant and comprises up to 29% of the total dry weight [3]. Despite their abundance, the polysaccharides of green macroalgae have not been well studied, compared to those of brown and red macroalgae. This could partly be due to limited industrial interest, the lack of detailed knowledge on their

structure and composition, and the lack of appropriate saccharification tools. Nevertheless, some progress has been made in recent studies reporting the discovery of enzymes that could depolymerise ulvan, together with details of the structure of ulvan [2,4–7]. However, a detailed characterisation of ulvan-depolymerising enzymes and the oligosaccharides they generate is still required. Ulvan is a water-soluble polysaccharide composed mainly of 3‑sulphated rhamnose (Rha3S), glucuronic acid (GlcA), iduronic acid (IdoA) and xylose (Xyl) [2]. The amounts of these individual sugars vary with the species, geographical location, time of harvest and the method of extraction [3,8–10]. The primary repeating disaccharide units of ulvan are Rha3S-GlcA (Type A), Rha3S-IdoA (Type B) and Rha3S-Xyl (Fig. 1). There are also minor repeating disaccharide units of sulphated and non-sulphated rhamnose (Rha3S-Rha3S, Rha3S-Rha or

⁎ Corresponding author at: Industrial Biotechnology, Department of Biology and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden. E-mail address: [email protected] (E. Albers).

https://doi.org/10.1016/j.algal.2018.10.016 Received 9 July 2018; Received in revised form 15 October 2018; Accepted 18 October 2018 2211-9264/ © 2018 Elsevier B.V. All rights reserved.

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Fig. 1. Ulvan and the action of ulvan lyase on ulvan. The blue dotted lines indicate potential sites for ulvan lyase action. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Due to the structural complexity of ulvan, several different enzymes are required for its depolymerisation. In Bacteroidetes and other Gramnegative microorganisms, the genes encoding these depolymerising enzymes, i.e., ulvan lyase, β‑glucuronyl hydrolase, α‑rhamnosidase, xylosidase, sugar-sensing and transporting proteins, are clustered into a genetic region called a polysaccharide utilisation locus (PUL). In F. agariphila, PUL 14, as assigned in the polysaccharide utilisation loci database, PULDB, contains several putative genes whose expression potentially confers the ulvan utilisation ability of the bacterium. Salinas and French confirmed PUL 14 as the ulvan utilisation loci through experimental and in silico analyses [20]. We recently reported a novel ulvan lyase (Cdf79930) from this ulvan utilisation loci [21]. Apart from Cdf79930, the ulvan utilisation loci express another putative ulvan lyase (Cdf79931). This putative ulvan lyase belongs to the PL28 family of the CAZy database. In this study, we describe the overexpression, purification and characterisation of the domains of the ulvan lyase from F. agariphila (FaUL), Cdf79931, and present the results of detailed analysis of the end products generated by its action on ulvan.

Rha-Rha). The presence of IdoA, a structural component of the mammalian glycosaminoglycans chondroitin sulphate, heparan sulphate and heparin, distinguishes ulvan from other marine polysaccharides. This distinctive characteristic makes ulvan a potential candidate for several biomedical and biotechnological applications [11]. Moreover, there has been an increasing interest in using this algal polysaccharide via biorefinery approaches to produce high-value products [12]. The depolymerisation of ulvan using enzymes would circumvent the use of harsh chemicals and energy intense mechanical processes used for the disintegration of the seaweed biomass. Polysaccharide lyases (PLs) catalyse the depolymerisation of polysaccharides containing uronic acids via β-elimination [13]. These polysaccharide lyases have been divided into 28 sequence-based families in the Carbohydrate-Active enZYmes (CAZy) database (www.cazy.org) [14]. In the depolymerisation of ulvan by ulvan lyases (EC.4.2.2), oligo-saccharides that contain an unsaturated uronic acid (∆) at the non-reducing ends are released (Fig. 1). Lahaye et al. first reported ulvan lyase activity in a marine bacterium isolated from mud containing decomposed ulvan in 1997 [4]. More than a decade later, an ulvan-utilising marine bacterium, Nonlabens ulvanivorans, belonging to the phylum Bacteroides, was identified, and an extracellular ulvan lyase was purified [6,15]. Several ulvan lyases that are similar to N. ulvanivorans lyase have subsequently been identified from members of the Alteromonadales order [5,16–18]. Analysis of the genome of a green macroalgae-associated flavobacterium, Formosa agariphila KMM3901, revealed the potential of this bacterium to utilise several marine algal polysaccharides, including ulvan [19]. Salinas and French further established the ulvan-utilisation capacity of this bacterium by growth studies using Ulva lactuca as the sole carbon source [20]. They also observed the induced expression of ulvan-depolymerising enzymes, including an ulvan lyase. Only six ulvan lyases have been characterised to date, from three polysaccharide lyase (PL) families: PL24, PL25 and PL28. Ulvan lyases of the Alteromonadales order belong to the PL24 and the PL25 families, while the ulvan lyases in N. ulvanivorans and F. agariphila belong to the PL28 family. PL24 family ulvan lyases exhibit specificity for the glycosidic bond between Rha3S and GlcA, while the remaining lyases in PL25 and PL28 act non-specifically on both Rha3S-GlcA and Rha3SIdoA.

2. Materials and methods 2.1. Ulvan Ulvan (winter heavy) from U. armoricana was obtained from Carbosynth (Compton, Berkshire, UK). Ulva lactuca was collected from Saltö (Strömstad, Sweden) in July, and U. intestinalis was collected at Tjörn close to Skärhamn (Sweden) in September, both from sites close to the water surface. The U. lactuca and U. intestinalis were identified based on the morphological structures [22]. Ulvan was extracted from these collected biomasses using a hot-water extraction method with ethanol washing and enzymatic purification, as described separately.1

1 Niklas Wahlström, Filip Nylander, Eric Malmhäll-Bah, Karin Sahlin, Ulrica Edlund, Gunnar Westman, Eva Albers, manuscript in preparation, unpublished observations.

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Fig. 2. In silico analyses of ulvan lyase. A. Graphical depiction of the domain structure of Cdf79931 and the recombinant constructs FaUL (~55 kDa), FaUL-CDBD (~47 kDa), FaUL-CD (~33 kDa). The yellow region indicates the 6xhistidine tag. B. Homology model and structural alignment of FaUL-CD with the ulvan lyase of N. ulvanivorans. The tertiary structure of FaUL-CD was modelled using the Phyre2 online tool and aligned with N. ulvanivorans 6D2C using UCSF-Chimera software. With the 68% sequence homology, the structures of the FaULCD and the N. ulvanivorans ulvan lyase are very similar. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

2.2. Bacterial strains and plasmid

FaUL-CD (additionally lacking the binding domain, amino acids from 11 to 275 of Cdf79931) were amplified from the F. agariphila genomic DNA using the primers listed in Table S1. The amplified genes were cloned into a modified pET28a(+) expression plasmid (with a TEV cleavage site replacing the thrombin site) between the Nde-I and Xho-I restriction sites to have an N-terminal 6xhistidine tag. The resulting recombinant plasmid was transformed into E. coli DH5α. The positive transformants with correctly oriented inserts were further transformed into E. coli BL21(DE3) for the expression studies. The overnight inoculum of E. coli BL21(DE3) harbouring the recombinant plasmid was transferred to a 2 L baffled Erlenmeyer flask containing 1 L of LB broth that contained 50 μg mL−1 kanamycin, and the culture was incubated at 37 °C. After the optical density (OD600) of the culture reached 0.8, the incubation was instead done at 16 °C for a further 30 min. After the 30 minute incubation, isopropyl β‑D‑1‑thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM to induce the recombinant protein expression and incubated for 16 h at 16 °C. The cells were harvested by centrifugation at 10,000 ×g for 10 min at 4 °C and resuspended in a lysis buffer (20 mM Tris buffer (pH 8.5), 500 mM NaCl). The cells were lysed by ultrasonication for 2 min with alternate cycles of 2 s on and 5 s off at 20% amplitude. The soluble fraction was collected by centrifugation at 12,000 ×g for 20 min, and loaded onto a 5 mL Histrap-Excel column (GE Healthcare Life Sciences, Chicago, IL, USA) attached to an Äkta purifier (GE Healthcare Life Sciences, USA) and pre-equilibrated with the lysis buffer. The unbound protein was washed with lysis buffer containing 60 mM imidazole, and the bound 6 × histidine-tagged recombinant protein was then eluted with elution buffer (20 mM Tris (pH 8.5), 500 mM NaCl, 500 mM imidazole). The collected eluted fractions were concentrated using a Centricon® concentrators with a membrane with a molecular weight cut-off of 10 kDa (Millipore Corp.,

Formosa agariphila KMM3901 (DSM No: 15362) was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) in freeze-dried form, and cultivated in Difco™ Marine Broth 2216 (3.74% w/v) at 25 °C and 200 rpm. Escherichia coli DH5α was used for gene cloning, and E. coli BL21(DE3) was used for the expression studies. Expression plasmid pET28a(+) was obtained from Novagen (Madison, WI, USA). 2.3. Bioinformatic analysis Predicted functional domains of Cdf79931 were identified using InterPro v68.0 (https://www.ebi.ac.uk/interpro/). The prediction of the presence of signal peptides using PredSi, SignalP and Phobius revealed a 10 amino acid length signal peptide that targets the protein to extracellular secretion [23–25]. The domains were further analysed using dbCAN web server tool available at DataBase for automated Carbohydrate-active enzyme Annotation [26]. The tertiary structure of the FaUL-CD was modelled using an online homology modelling tool - Phyre2 and the resulting 3D-structure was analysed using Verify3D and PROCHECK for the quality [27–29]. DALI server was used to find the structural homologs of the FaUL-CD [30]. 2.4. Cloning, expression and purification F. agariphila genomic DNA was isolated using the DNeasy® Plant Mini Kit (Qiagen GmbH, Hilden, Germany). The gene regions that encode the F. agariphila ulvan lyase (FaUL, lacking signal peptide, amino acids from 11 to 500 of Cdf79931), FaUL-CDBD (lacking signal peptide and sorting domain, amino acids from 11 to 405 of Cdf79931), and 108

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microplate reader (BMG LABTECH, Germany) set at 232 nm. 2.6.2. Optimum pH and temperature The optimum pH and temperature of the FaUL were determined by incubating 2 μg of the enzyme with 200 μg of ulvan extracted from U. armoricana. For pH curve, 0.2 mL of 100 mM citrate-phosphate buffer (pH of 5.5, 6, 6.5 and 7), 100 mM phosphate buffer (pH of 6.5, 7, and 7.5), 100 mM Tris-HCl buffer (pH of 7.5, 8, 8.5 and 9), or 100 mM Glycine-NaOH buffer (pH of 8.8, 9, 9.5 and 10) were used. For the temperature curve, the ulvan assay was carried out between 20 and 60 °C. 2.7. Enzyme kinetics measurements Two micrograms of FaUL (lacking N-terminal signal peptide) and FaUL-CD (only the catalytic domain) were incubated with varying concentrations of ulvan (0.25–2 mg mL−1) extracted using a hot-water extraction method with ethanol washing and enzymatic purification1 from three different species: U. armoricana, U. intestinalis and U. lactuca, and the initial velocities were determined by measuring the absorbance at 232 nm. The initial velocity was plotted against the substrate concentration, and the resulting graph was fitted to the Michaelis-Menten kinetics equation by non-linear regression using GraphPad® Prism® software. Fig. 3. Purification of ulvan lyase. A. Size exclusion chromatography of FaUL (solid line), FaUL-CDBD (dashed line) and FaUL-CD (dotted line). Ulvan lyase activity was associated with the major peaks at ~100 mL. B. SDS-PAGE analysis of the purified samples. The protein bands in the stain-free Bio-Rad® gel were visualised using a Chemidoc®. Lane M, protein molecular weight markers; Lane 1, FaUL-CD (~33 kDa) purified with size exclusion chromatography; Lane 2, FaUL-CD eluted from the immobilised metal affinity column; Lane 3, FaULCDBD (~47 kDa) purified with size exclusion chromatography; Lane 4, FaULCDBD eluted from the immobilised metal affinity column; Lane 5, FaUL (~55 kDa) purified with size exclusion chromatography; Lane 6, FaUL eluted from the immobilised metal affinity column.

2.8. Separation of oligosaccharides generated by FaUL action on ulvan Five milligrams of ulvan extracted from U. lactuca dissolved in 0.5 mL of 20 mM Tris buffer (pH 8.5) with 150 mM NaCl was mixed with 50 μg of the recombinant FaUL and incubated at 37 °C for 12 h, after which the lysed mixture was boiled for 5 min to stop the reaction. The samples were then centrifuged to remove the protein precipitate, and the supernatant was loaded onto HiPrep™ 26/60 Sephacryl® S-100 HR and Biogel-P2 (100 cm × 1.5 cm) columns connected in series to the Äkta Explorer instrument (GE Healthcare Life Sciences, USA). Separation was carried out using 50 mM ammonium carbonate buffer (pH 8.0) as the eluent and was monitored with a UV detector operating at 232 nm. The fractions that corresponded to each peak were pooled, freeze-dried, and stored at 4 °C until further analysis.

Burlington, MA, USA). Each concentrated sample was loaded onto a HiPrep™ 16/60 Sephacryl S-100 HR size-exclusion column (GE Healthcare Life Sciences), which had been equilibrated with a buffer containing 20 mM Tris HCl (pH 8.5) and 150 mM NaCl. The eluted protein was collected into fractions. However, only the fractions that displayed the ulvan lyase activity were concentrated using a Centricon® concentrator with a 10 kDa cut-off membrane, as above, and analysed on a TGX Stain-Free gel (4%–15% acrylamide; Bio-Rad Laboratories, Hercules, CA, USA).

2.9. NMR analysis of ulvan oligosaccharides generated by FaUL The separated end products were dissolved in D2O (99.97% atomic deuterium), and their structures determined using 1H NMR and 13C NMR. The 1H NMR and 13C NMR spectra were acquired on a Bruker Avance III HD NMR spectrometer equipped with a TXO cryogenic probe, operating at 800 MHz frequency, using the solvent residual signal as the chemical shift reference.

2.5. Enzyme assays The enzyme activity measurements and characterisation studies were performed using the purified enzyme samples obtained by sizeexclusion chromatography. The ulvan lyase activity was initiated by mixing 2 μg of the enzyme with 200 μg of ulvan in 0.2 mL of 20 mM Tris-HCl pH 8.5, and the activity was determined by measuring the formation of unsaturated oligosaccharide products in the assay mixture through the increase in absorption at 232 nm.

2.10. Mass spectrometry of ulvan oligosaccharides generated by FaUL Before analysis, the separated ulvan oligosaccharide samples were reduced by treatment with 0.5 M NaBH4 and 20 mM NaOH at 50 °C overnight. The samples were then desalted using a cation-exchange resin (AG® 50W-X8 Cation Exchange Resin: Bio-Rad) packed onto a ZipTip C18 tip (Millipore® Corp, Burlington, MA, USA). After drying in a SpeedVac (Thermo Scientific™ Savant™), methanol was added to remove the residual borate by evaporation. The resultant oligosaccharides were dissolved in Milli-Q water at the same volume as the starting material (~50 μL) and then analysed using LC-MS/MS in negative-ion mode. The oligosaccharides were separated on a column (10 cm × 250 μm) packed in-house with 5 μm porous graphite particles (Hypercarb; Thermo-Hypersil, Runcorn, Cheshire, UK). The oligosaccharides were injected into the column and eluted with an acetonitrile gradient (Buffer A: 10 mM ammonium bicarbonate; Buffer B: 10 mM ammonium bicarbonate in 80% acetonitrile). The gradient

2.6. Enzyme characterisation 2.6.1. Substrate specificity FaUL and FaUL-CD were tested for their substrate specificities. 2 μg of protein was mixed with 200 μg of chondroitin sulphate, heparan sulphate, xanthan, pectin or alginate added in 0.2 mL of 20 mM Tris buffer (pH 8.5) and incubated at 37 °C. Appropriate controls, i.e., buffer, enzyme, and substrate blanks, were treated similarly. The progress of the enzyme action was monitored using a FLUOstar Omega 109

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Fig. 4. The action of ulvan lyase on ulvan. A. The Michaelis–Menten constant (Km) of ulvan extracted from three different Ulva species. B. The separation of the end products from the action of FaUL on ulvan from U. lactuca. The ulvan oligosaccharide end products were separated on an Äkta Explorer connected with HiPrep™ 26/ 60 Sephacryl® S-100 HR and Biogel-P2 (100 cm × 1.5 cm) columns in series. Separation was carried out using 50 mM ammonium carbonate buffer (pH 8.0) as the eluent, and the elution was monitored with a UV detector operating at 232 nm. ERF = enzyme-resistant fraction; C. Degradation time course of FaUL acting on ulvan from U. lactuca. 50 μg of FaUL was incubated with 5 mg of ulvan from U. lactuca at 37 °C. The time-course of ulvan depolymerisation by FaUL shows the initial generation of the larger unsaturated fragments, which disappear with the increase in incubation time due to the endolytic action of the FaUL to form the smaller ulvan oligosaccharides, and finally yields the di-, tri-, tetra- and pentasaccharides as the end products.

(0%–45% Buffer B) was eluted for 46 min, followed by washing with 100% Buffer B, and equilibration with Buffer A for 24 min. A 40 cm × 50 μm i.d. fused silica capillary was used as the transfer line to the ion source. The samples were analysed in negative-ion mode on an LTQ linear ion trap mass spectrometer (Thermo Electron, San José, CA, USA), with an IonMax standard ESI source equipped with a stainlesssteel needle maintained at −3.5 kV. Compressed air was used as the nebulizer gas. The heated capillary was maintained at 270 °C, and the capillary voltage was −50 kV. A full scan (m/z 380–2000, two microscans, maximum 100 ms, target value of 30,000) was performed, followed by data-dependent MS2 scans (two microscans, maximum 100 ms, target value of 10,000) with a normalized collision energy of 35%, an isolation window of 2.5 units, activation q of 0.25, and an activation time of 30 ms. The threshold for MS2 was set to 300 counts. Xcalibur ver. 2.0.7 software was used for acquisition and processing.

typically cleaved from the protein before secretion into the extracellular milieu [31]. Three constructs were made, FaUL (lacking N-terminal signal peptide), FaUL-CDBD (lacking both the N-terminal signal peptide and C-terminal sorting domain), and FaUL-CD (only the catalytic domain), see Fig. 2A. More than 95% of residues of FaUL-CD were homology modelled at > 90% confidence, and the resulting 3D-structure is displayed in Fig. 2B in comparison to the N. ulvanivorans ulvan lyase (PDB id 6D2C). Additionally, the structural similarity search using the DALI server revealed the structural neighbours that are majorly alginate lyases (PDB id - 3ZPY, 4BE3, 5ZU5, 1J1T, 5XNR, and 4Q8K). 3.2. Heterologous expression, purification and biochemical characterisation The three different constructs of the Cdf79931 (FaUL, FaUL-CDBD and FaUL-CD) were expressed in E. coli (DE3), and the expressed proteins were in the soluble fraction when induced at 16 °C. The Ni-affinity-purified recombinant proteins were all eluted by size-exclusion chromatography as a single major peak (Fig. 3A and B). The FaUL and FaUL-CD were highly expressed and yielded 38 mg and 80 mg of the recombinant protein per litre of the culture, respectively, whereas the expression of FaUL-CDBD was only 8 mg per litre of the culture. The curve of initial velocity versus pH was bell-shaped, and the maximum initial velocity was observed at pH 8.5 (Supplementary Fig. 1). It exhibited a high tolerance to salt, and a significant increase in activity was observed in the presence of NaCl at concentrations up to 500 mM (Supplementary Fig. 1). However, the activity decreased above 500 mM NaCl. FaUL and FaUL-CD could not depolymerise the chondroitin sulphate,

3. Results 3.1. In silico analyses Signal peptide analysis of the Cdf79931 amino acid sequence using SignalP and PrediSi indicated the presence of a 10-amino-acid signal peptide. The InterPro and BLASTp analyses of the Cdf79931 sequence revealed the presence of a catalytic domain followed by a B-lectin-like domain and a Por secretion system C-terminal sorting domain (Fig. 2A). Further analysis of the B-lectin domain using dbCAN revealed it as a Carbohydrate Binding Module 13. Por secretion system C-terminal sorting domain protein is a part of the type IX secretion system, and it is 110

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Fig. 5. LC-MS analysis of the end products of U. lactuca ulvan depolymerisation by FaUL separated by size-exclusion chromatography presented in Fig. 4B: Fraction 1 (A), Fraction 2 (B) and Fraction 3 (C). The larger oligosaccharide species eluted first with the major pentasaccharide and tetrasaccharide fragments eluting in Fraction 1, and a mix of tetrasaccharide and trisaccharide (Rha3S-Xyl-Rha3S) and unsaturated disaccharide (∆Rha3S) fragments majorly eluting in Fraction 2 and 3 respectively. Fig. 6. Branching in U. lactuca ulvan. LC-MS/MS analysis of a pentasaccharide (m/z-839) indicates that the pentasaccharide contains a branched unsaturated hexuronic acid. The unsaturation of the branched uronic acid residue is a result of the lysis of the branch by FaUL. So, the unsaturated uronic acid residue at the branching of the ulvan indicates that the branch is longer than the single residue.

heparan sulphate, pectin, xanthan or alginate. The FaUL was active between 20 and 50 °C and displayed no activity at 60 °C (Supplementary Fig. 1). FaUL exhibited a Km of 0.70 ± 0.02 mg mL−1, 3.0 ± 0.1 mg mL−1, and 1.20 ± 0.03 mg mL−1 when incubated with ulvan isolated from U. armoricana, U. intestinalis and U. lactuca, respectively (Fig. 4A). FaUL-CD exhibited a Km of 0.60 ± 0.03 mg mL−1, 2.6 ± 0.1 mg mL−1, and 0.70 ± 0.02 mg mL−1 when incubated with ulvan isolated from U. armoricana, U. intestinalis and U. lactuca, respectively (Fig. 4A).

length of these large oligosaccharide fragments decreased with the extension of the incubation time. After 12 h of incubation with a two-fold of the enzyme, the ulvan was depolymerised entirely to small oligosaccharides (Fig. 4B and C). These oligosaccharide end products were separated by BioGel-P2 size-exclusion chromatography and collected into three different fractions (Fig. 4B). The oligosaccharides in these fractions were analysed with 1H NMR and mass spectrometry. Mass spectrometry revealed that the fractions collected during the size-exclusion chromatography were not pure but were instead a mixture of oligosaccharides (Fig. 5). Fraction 1 (Kav = 0.29) consisted predominantly of a mixture of the tetrasaccharide ∆Rha3S-Xyl-Rha3S and the pentasaccharides Rha3S-XylRha3S-Xyl-Rha3S and ∆Rha3S-Xyl-(∆)Rha3S (Fig. 5A). Further fragmentation analysis of the pentasaccharide ∆Rha3S-Xyl-(∆)Rha3S (m/z 839) peak using LC-MS/MS indicated branching on the terminal rhamnose (Fig. 6). The branching residue was an unsaturated hexuronic acid. This is the first evidence of longer branching in ulvan otherwise known to have single residue branches as observed by Lahaye and Ray [35]. Fraction 2 (Kav = 0.34) contained mainly the tetrasaccharides

3.3. Separation and analysis of the end products of the ulvan breakdown The time course of depolymerisation of ulvan from U. lactuca by FaUL was monitored by measuring the absorption at 232 nm, which is typical for eC]Ce unsaturation in oligosaccharides, separated on a size-exclusion chromatography column. Initially, there was an accumulation of long chains of ulvan with unsaturated non-reducing ends that were eluted out of the size exclusion column very early. This indicates that the FaUL depolymerised ulvan in an endolytic fashion and generated large fragments. The 111

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monosulphated xylose-containing trisaccharide (Rha3S-Xyl-Rha) was the major end product of FaUL action (Fig. 8A). Depolymerisation of the ulvan of U. armoricana by FaUL yielded very few oligosaccharides, whereas ulvan of U. intestinalis and U. lactuca yielded several different xylose-containing oligosaccharides, which eluted late on the porous graphite carbon column during LC-MS (Fig. 8B and C). 4. Discussion Ulvan is one of the most complex and sulphated polysaccharides. Due to its complexity, the depolymerisation of ulvan requires several enzymes that act at different degrees of polymerisation and different subcellular locations. The genome of Formosa agariphila revealed the potential of this bacterium to utilise several algal polysaccharides, and the presence of a PUL for ulvan [19]. The BN863_22190 locus of this PUL encodes a putative ulvan lyase, Cdf79931. Salinas and French [20] have confirmed the product of this putative gene to be an extracellular ulvan lyase. In the present study, we overexpressed and characterised this extracellular ulvan lyase, which plays a crucial role in the initial depolymerisation of the ulvan polysaccharide. Two different truncations of this protein that lack N-terminal signal peptide were also heterologously overexpressed in E. coli. In contrast to the previous observations on ulvan lyase of N. ulvanivorans [6,32], a full length construct with C-terminal sorting domain (FaUL) was overexpressed without any problems of degradation or accumulation as inclusion bodies. The predicted tertiary structure of the FaUL-CD displayed β-jelly roll fold and showed high similarity to the structure of N. ulvanivorans ulvan lyase (Fig. 2). This fold is also found in PL7, PL13, PL14, PL18, PL20, and PL28 families on CAZy database. The search for the structural homologs on DALI server revealed the alginate lyases (PDB id 3ZPY, 4BE3, 5ZU5, 1J1T), glucuronan lyase (PDB id - 2ZZJ) and heparin lyase (PDB id - 3IKW) as the closest structural homologs after the N. ulvanivorans ulvan lyase. Based on the structural homology, the conserved residues Tyr244, Gln125, and Lys127 could be the potential catalytic residues. Further studies involving site-directed mutagenesis of these residues will reveal the mechanistic properties of the FaUL action. All the constructs were catalytically active and could depolymerise the ulvan. Mass spectrometry and 1H NMR analyses indicated that the end products of FaUL action were ∆Rha3S, Rha3S-Xyl-Rha3S, ∆Rha3S-XylRha3S, Rha3S-Xyl-Rha3S-Xyl-Rha3S and branched ∆Rha3S-Rha3S-Xyl-(∆) Rha3S. In similarity to the previously studied ulvan lyases, the reducing ends of the principal end products of FaUL terminated with Rha3S, and non-reducing ends with 4‑deoxy‑L‑threo‑hex‑4‑enopyranosiduronic acid, which is formed irrespective of the uronic acid partner in the parent polysaccharide [6]. FaUL-CD exhibited higher affinity for the ulvan than the full-length construct (FaUL) that has the ulvan binding domain. The role of the ulvan binding domain in the function of ulvan lyase hasn't been established yet. Melcher et al. proposed that the binding domain may help in accessing the semi-crystalline insoluble substrate as present in nature [32]. The full-length FaUL exhibited slightly higher apparent Km (0.72 ± 0.02 mg mL−1 for ulvan from U. armoricana) than that of the ulvan lyase from N. ulvanivorans, with Km reported to be 0.5 mg mL−1 of ulvan from U. rotundata [6]. The Km values of the ulvan lyases isolated from Pseudoalteromonas (2.1 mg mL−1, unknown source), Glaciecola (4.1 mg mL−1 of ulvan from U. ohnoi), and Alteromonas (7.2 mg mL−1 of ulvan from U. ohnoi) are much higher than that of FaUL [17,33,34]. These differences in kinetic properties could be due to the composition of the substrates used. Ulvan lyases from Glaciecola and Alteromonas show high specificity towards the β‑(1,4) glycosidic bond in Rha3S-GlcA in ulvan. This specificity could have contributed to the higher Km. Both FaUL and FaULCD exhibited higher Km with the ulvan extracted from U. intestinalis. This could be due to the high amounts of Rha3S-Xyl repeating disaccharide units in the ulvan of U. intestinalis. FaUL was optimally active at pH 8.5, consistent with the mild alkaline conditions of shallow-water, the natural

Fig. 7. 1H NMR analysis of the end products of U. lactuca ulvan depolymerisation by FaUL separated with size-exclusion chromatography of Fig. 4B: Fraction 1 (A), Fraction 2 (B) and Fraction 3 (C). The peaks were assigned based on the spectra reported by Lahaye et al. [14,26]. Two prominent signals at 5.97 ppm (∆H1) and 5.4 ppm (∆H4), indicate the unsaturation generated due to the ulvan lyase activity, appeared in all the oligosaccharide fractions collected from size-exclusion chromatography.

∆Rha3S-Xyl-Rha3S and ∆Rha3S-Xyl-Rha, and the trisaccharide Rha3SXyl-Rha3S (Fig. 4B). Fraction 3 (Kav = 0.4) contained mainly a disaccharide (approximately 90%), a minor proportion of Rha3S-XylRha3S (Fig. 5C). Further analysis of these di- and trisaccharides through fragmentation with LC-MS/MS confirmed that they were an unsaturated hexopyranosiduronic acid linked to Rha3S (∆Rha3S), and a trisaccharide that contains a xylose with flanking Rha3S on both sides (Rha3S-Xyl-Rha3S). The separated oligosaccharide end products were further incubated with excess FaUL for 12 h. However, no further degradation of these oligosaccharides was observed. The 1H NMR spectra of the ulvan oligosaccharide end products were compared to the spectra reported by Lahaye et al. [14,26], and peak shifts were assigned (Fig. 7). Two prominent signals at 5.97 ppm (∆H1) and 5.4 ppm (∆H4), which are characteristic of ulvan lyase activity, appeared in all the oligosaccharide fractions collected from size-exclusion chromatography, indicating that the oligosaccharides have an unsaturated bond at the non-reducing end. As the composition of ulvan changes with the species, FaUL action was tested with the ulvan extracted from three different sources, U. armoricana, U. intestinalis and U. lactuca. The FaUL was incubated with the substrate for 12 h, and the resulting mixture of oligosaccharides were analysed directly on LC-MS. For all the three ulvans tested, a 112

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Fig. 8. The ion chromatograms from LC-MS analysis of the end products resulting from the action of FaUL for 12 h on ulvan from: U. armoricana (A), U. intestinalis (B), and U. lactuca (C). The elution profile of the three samples from the porous graphite carbon column is different. Depolymerisation of the ulvan of U. armoricana by FaUL yielded a very few oligosaccharides whereas ulvan of U. intestinalis and U. lactuca yielded several different xylose-containing oligosaccharides, which eluted late on during the LC-MS analysis. In all the three depolymerised ulvan samples, a monosulphated xylose-containing trisaccharide (Rha3S-XylRha) was the major end product of FaUL action.

habitat of the marine bacterium Formosa agariphila [19]. Similar to another PL28 ulvan lyase from N. ulvanivorans, FaUL was optimally active at 45 °C and displayed decent activity at broad-range of temperatures from 20 to 50 °C [6]. The ulvan depolymerisation activity at the ambient temperatures is particularly advantageous in the biorefinery processes as it avoids the high energy consumption required for heating the reaction mixture. The optimal activity of FaUL in the presence of 0.25 M NaCl represents the possible adaptation of the host bacterium to the moderate saline environment of the coastal shallow-waters. The composition of the ulvan varies among the Ulva species [3,9]. The oligosaccharide profiles of the ulvans from the three different Ulva species significantly differed in the levels of xylose-containing oligosaccharides after the depolymerisation by FaUL. The lower levels of xylose-containing oligosaccharides in U. lactuca and U. armoricana ulvan samples could be due to lower levels of xylose [4]. High levels of xylose-containing oligosaccharides in ulvan of U. intestinalis could reflect higher levels of xylose. This could have also contributed to the higher Km of the FaUL and FaUL-CD with this substrate as the ulvan lyases cannot act on the glycosidic bond between rhamnose and xylose. Very few details are known regarding the branching in ulvan [2,35]. Surprisingly, the branches in ulvan were only a single residue length, and there hasn't been any evidence for the length of the branches beyond this first uronic acid residue [35]. In this study, we found an unsaturated uronic acid linked to the O2 of the non-sulphated rhamnose in enzyme-degraded ulvan oligosaccharides. This unsaturated uronic acid was generated due to the lysis of the glycosidic bond between its partner rhamnose residue/branch by FaUL. With this, we provide evidence for the possibility of the extension of the branch beyond the first branched uronic acid residue. Lahaye et al. [35] also observed branching in ulvan at the O2 position. However, no details regarding the frequency and lengths of the branches are currently known. Further studies on the branching of ulvan would help to explain its low viscosity and other physicochemical properties. The branching of ulvan may also influence the binding of the substrate and, thus, the Km. The availability of characterised ulvan-depolymerising enzymes,

such as the ulvan lyase investigated in this study, is a major requirement in the biorefinery of biomass of different Ulva species. In this study, we report the overexpression and characterisation of the ulvan lyase of Formosa agariphila and the detailed analysis of its ulvan depolymerising activity. This lyase could be useful in generating the ulvan oligomers that have potential applications in medicine as drug-delivering, anti-coagulating and anti-viral agents, and applications in agriculture as plant growth promoters [11,36,37]. The enzymatic depolymerisation of the ulvan and subsequent saccharification to fermentable sugars brings forward the alternative potential applications of green tides-biomass in the production of biofuels, such as ethanol and butanol via clostridial fermentation. In this study, we tested ulvan lyase applicability in the depolymerisation of ulvan extracted from three different Ulva species. The composition of Ulva biomass may vary with the season and geographical location. Additionally, variations in the dynamics and thermochemical properties of the water can cause differences in the amount of the ulvan and its chemical composition, such as sulfonation and individual monosaccharide content. The composition of ulvan in the Ulva biomass obtained from the eutrophicated waters may be different from the ulvan tested in this study. Hence, further studies involving the Ulva biomass of algal blooms of eutrophicated waters are required to further establish the methods for the Ulva biorefinery. Acknowledgements We thank our colleagues Joakim Olsson, Chalmers, and Göran Nylund and Friedrike Eimer at Gothenburg University for help with the collection of U. lactuca and U. intestinalis biomass, and Eric MalmhällBah at our division for assistance with ulvan extraction of these. We acknowledge the Swedish NMR Centre at Gothenburg University for carrying out the NMR analyses of the end-products.Funding This study was supported by the Swedish Foundation for Strategic Research (SSF), Sweden, (RBP14-0045) to EA. 113

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Author contributions and agreements V.R.K. designed and performed all the experiments except the LCMS and NMR spectra measurements. E.A. secured the funding of the project, aided in the design of the experiments and discussed results with V.R.K. C.J. performed the LC-MS analyses while N.G.K. provided the resources for these analyses. V.R.K. wrote the manuscript with the contribution from C.J. for LC-MS experimental part. E.A., V.R.K., and C.J. reviewed the manuscript. All authors agree to authorship and submission of this manuscript.

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Declarations of interest

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The authors have no conflicting interests to declare. No conflicts, informed consent, human or animal rights are applicable.

[19]

[15] [16] [17]

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Appendix A. Supplementary data

[21]

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.algal.2018.10.016.

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References

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