Cloning and characterization of the first polysaccharide lyase family 6 oligoalginate lyase from marine Shewanella sp. Kz7

Abstract

Alginate, the most abundant carbohydrate in brown macroalgae, is widely used in the food and pharmaceutical industries. Recently, alginate has attracted increasing attention, as it may serve as an alternative biomass for the production of biofuel. The degradation of alginate into monomeric units is the prerequisite for bioethanol production. All known oligoalginate lyases belong to the polysaccharide lyase (PL) family 7, 14, 15 and 17, and most of them preferred to degrade the polyM blocks to yield 4-deoxy-l-erythro-5-hexoseulose uronic acid as the primary product. In this study, we cloned an oligoalginate lyase gene, oalS6, from Shewanella sp. Kz7 and expressed it in Escherichia coli. The PL family 6 oligoalginate lyase (OalS6) has no significant sequence similarity with other known oligoalginate lyases. OalS6 contains a chondroitinase-like domain and was assigned to the PL family 6. This lyase is an exo-type oligoalginate lyase and prefer to depolymerize polyG block into 2, 4, 5, 6-tetrahydroxytetrahydro-2H-pyran-2-carboxylic acid. All of these results indicate that OalS6 is a novel oligoalginate lyase that is structurally and functionally different from other known oligoalginate lyases. This finding provides new insights into the development of biofuel processing biotechnologies from seaweed.

Alginate, an acidic heteropolysaccharide, consists of β-d-mannuronate (M) and α-l-guluronate (G), which arrange into polyM, polyG, and heteropolymeric random polyMG blocks (1). Alginate is the most abundant carbohydrate in brown algae and commercially useful due to its high viscosity and gelling property. Besides, some bacteria, such as Pseudomonas aeruginosa and Azotobacter vinelandii, can also synthesize alginates (2). Since world energy demand continues to rise and fossil fuel resources are depleted, the application of alginate for bioethanol production has received increasing attention (3, 4).

Alginate lyase is an enzyme that catalyses the degradation of alginate with a β-elimination mechanism. It introduces a double bond between C4 and C5 at the non-reducing end (5). The enzymatic degradation products have been found to exhibit a variety of bioactive functions (6, 7). Alginate lyases also used in the protoplast production of brown algae, and in the application of improving antibiotic killing of mucoid P. aeruginosa in biofilms (8, 9). Alginate lyases from different sources have been assigned into 7 polysaccharide lyase (PL) families, namely PL-5, 6, 7, 14, 15, 17 and 18, as shown in the carbohydrate-active enzymes (CAZy) database (http://www.cazy.org/). The mode of action of alginate lyase can be further classified into endo- and exo-lytic mechanisms (5). The majority of alginate lyases have been documented as endo-type lyases, degrading alginate into unsaturated di-, tri- and tetra-saccharides as the main products (5), whereas exo-type alginate lyase (oligoalginate lyase) degrade both alginate polymer and oligosaccharide into unsaturated monomers (Supplementary Fig. S1). The unsaturated monosaccharide is non-enzymatically transformed into 4-deoxy-l-erythro-5- hexoseulose uronic acid (DEH), which was then reduced to 2-keto-3-deoxygluconate (KDG) by a reductase DehR and fed into the Entner–Doudoroff (ED) pathway to produce ethanol (3, 4). Genes encoding alginate-related metabolic enzymes and transport systems have been transferred into Escherichia coli, making the engineered bacterium able to produce ethanol from alginate (3). Recently, an efficient ethanol production method using the alginate monosaccharides as the raw material has been established in Saccharomyces cerevisiae (4). Therefore, the degradation of alginate into monomeric units is the prerequisite for bioethanol production (10–12).

Thus far, only few oligoalginate lyases have been experimentally characterized as exo-type lyases producing alginate monosaccharides as the main products (10–17). Oligoalginate lyases from different sources have been assigned into four PL families in the CAZy database, including PL-15 oligoalginate lyases A1-IV from Sphingomonas sp. A1, Atu3025 from Agrobacterium tumefaciensstrain C58 and AlyA from Vibrio splendidus; PL-17 oligoalginate lyases Alg17C from Saccharophagus degradans 2-40, AlgL from Sphingomonas sp. MJ-3, OalS17 from Shewanella sp. Kz7, OalB and OalC from V. splendidus; AlyA5 in PL-7 from Zobellia galactanivorans, HdAlex in PL-14 from Haliotis discus hannai (10–17). To date, the reported oligoalginate lyases were predominantly polyM block preferred enzymes.

In this study, we cloned a PL family 6 oligoalginate lyase (OalS6)-encoding gene from a marine bacterium, Shewanella sp. Kz7, expressed it in E. coli, then purified and characterized the recombinant lyase. OalS6 was a polyG preferred oligoalginate lyase, which could be used in combination with polyM preferred enzymes for monosaccharide production from alginate.

Materials and Methods

Bacterial strains and culture conditions

Strain Kz7 (CCTCC No. AB2014040) is now preserved in the China Center for Type Culture Collection, and used as the source strain of the oligoalginate lyase gene. It was cultured at 25°C in an alginate minimal medium (3 g sodium alginate, 3 g KH₂PO₄, 7 g K₂HPO₄·3H₂O, 2 g (NH₄)₂SO₄, 30 g NaCl, 0.05 g FeSO₄·7H₂O, 0.01 g MgSO₄·7H₂O, 2 g peptone, 15 g agar in 1 l distilled water, pH 7.0). Escherichia coli strains DH5α and BL21(DE3) were grown at 37°C in Luria-Bertani (LB) broth supplemented with either ampicillin (50 µg/ml) or kanamycin (30 µg/ml) if necessary. The oligonucleotides used in this study are shown in Supplementary Table SI.

Isolation of OalS6

Degenerate primers (AlgF and AlgR) were designed according to the conserved amino acid sequences of the alginate lyases in the PL-6 family; using these primers, the central part of OalS6-encoding gene (1,355 bp in length) was amplified from the genomic DNA of Shewanella sp. Kz7 and sequenced. The flanking sequences were isolated with SiteFinding-PCR and nine nested specific primers (SFP1-3, Up-OalS6-sf-asF1-3, Down-OalS6-sf-sF1-3) (18). The PCR products were purified, sequenced and assembled with the central portion, yielding the full-length gene sequence. The ORF was identified with the DNATools program. The signal peptide was predicted using the SignalP 3.0 server (http://www.cbs.dtu.dk/services/SignalP-3.0/). The theoretical molecular weight (Mw) was calculated using the Compute Mw Tool (http://us.expasy.org/tools/). Multiple sequence alignment was obtained with the ClustalX program. The conserved domains of OalS6 and other alginate lyases were searched with InterProScan 4 running the HMMPfam application (http://www.ebi.ac.uk/Tools/pfa/iprscan/).

Expression and purification of recombinant OalS6

The primers (OalS6-EF and OalS6-ER) possessed NdeI and XhoI sites at their 5′-ends. The PCR product was digested with NdeI and XhoI, and ligated into the pET-28 a (+) vector previously digested with NdeI and XhoI. The recombinant plasmid, pET28-oalS6, was transferred into E. coli BL21(DE3). Protein expression was induced at OD₆₀₀ of 0.6 with 0.5 mM isopropyl-β-thiogalactoside for 6 h at 25°C and 120 revolutions per minute. The bacterial cells were harvested and sonicated in the lysis buffer [20 mM phosphate buffer (pH 7.0), 500 mM NaCl, 10 mM imidazole]. Then the soluble fraction of protein was obtained by centrifugation at 10,000 g at 4°C for 30 min, and loaded on Ni-Sepharose column. The column was washed with the wash buffer [20 mM phosphate buffer (pH 7.0), 500 mM NaCl, 50 mM imidazole], and the target protein was eluted with elution buffer [20 mM phosphate buffer (pH 7.0), 500 mM NaCl, 150 mM imidazole]. Then the elution fraction was desalted using a disposable PD-10 desalting column (GE Healthcare) with 20 mM phosphate buffer (pH 7.0) as the mobile phase. The Mw of OalS6 was determined with sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE). The protein concentration was determined using the method of Lowry et al. (19) using bovine serum albumin as the standard.

Enzymatic activity assay

One hundred microliters of enzyme solution (in 20 mM phosphate buffer, pH 7.0) was mixed with 900 μl of substrate solution [0.3% (w/v) in 20 mM phosphate buffer, pH 7.0] and incubated at 40°C for 10 min. The relative enzyme activity was measured using the 3, 5-dinitrosalicylic acid method (10, 14, 20) with glucuronic acid as the standard. One unit was defined as the amount of enzyme causing the release of 1 μmol reducing sugar per minute under the above conditions.

Effect of temperature, pH, chelators and metal ions on OalS6 activity

The optimum temperature for OalS6 activity was determined in 20 mM sodium phosphate buffer, pH 7.0, at various temperatures from 0 to 60°C. The thermostability of OalS6 was evaluated by measuring the residual activity of the enzyme after incubation at different temperatures for 1 h. The effect of pH on alginate activity was determined under the standard assay conditions at 50°C in various buffers (50 mM): citrate buffer (pH 4.0–6.0), phosphate buffer (pH 6.0–8.0), Tris-HCl buffer (pH 7.5–9.0) and glycine-NaOH buffer (pH 8.6–10.6). To determine the pH stability of OalS6, the residual enzyme activity was measured after incubation at 4°C in various pH buffers (pH 4.0–10.6) for 24 h. The effect of metal ions and chelators on OalS6 activity was examined by monitoring its enzymatic activity in the presence of various cations or chelators. Comparisons were made by Student’s t

test using SPSS 19.0.

Analysis of substrate specificity and kinetic parameters

Sodium alginate and polyM and polyG block [0.3% (w/v) in 20 mM phosphate buffer, pH 7.0] were digested with OalS6 to determine its substrate specificity at 40°C for 10 min. The initial velocities (v₀) were determined by measuring the activity at 3–6 different time points. The kinetic constants were obtained from at least triplicate measurements at varying concentrations of alginate (from 0.1 to 8 mg/ml of substrate). The enzymatic activity was determined using the standard assay conditions, except that the reaction time was 3 min. The apparent kinetic parameters, including Km, Vmax and kcat/Km, were obtained from Lineweaver–Burk plots, which were assessed using a standard linear regression function.

Determination of the action mode of OalS6

The action mode of OalS6 was examined using polyG block and disaccharide as the substrates. The reaction products were also analysed using an ÄKTA FPLC (GE Health) with a superdex peptide 10/300 GE column (GE Health) at 235 nm, in which the mobile phase was 0.2 M ammonium bicarbonate (0.3 ml/min).

Determination of alginate disaccharides degradation of OalS6

The alginate disaccharide was prepared from the degradation products of alginate by AlyV5 from Vibrio sp. QY105 (21) and purified using a Biogel P-6 column. The degradations of alginate-derived disaccharides (10 mg/ml) by OalS6 (0.1 mg/ml) were analysed using TLC chromatography at different reaction times (0, 30, 60 min). Reaction products were visualized on a HPTLC plate developed with n-butanol/formic acid/water (2:1:1, by vol) and they were visualized with a diphenylamine/aniline/phosphate reagent (22). Then, the reaction products were further analysed by FPLC with a superdex peptide 10/300 GE column (GE Health) at 235 nm, in which the mobile phase was 0.2 M ammonium bicarbonate (0.3 ml/min).

Analysis of reaction products

The enzyme (0.5 mg) was added to 10 ml of polyG block (3 mg/mL in 20 mM phosphate buffer, pH 7.0) and incubated at 40°C until the substrate was degraded completely. To identify the structure of the reaction products, the main products were purified by TLC on silica gel plates (20 × 20 cm, GF254, Qingdao Marine Chemical Factory), then analysed using negative ion electrospray ionization mass spectrometry (ESI-MS), ¹H-NMR, ¹³C-NMR and 2D ¹H-¹H COSY NMR spectroscopy, as described previously (23, 24).

Nucleotide sequence accession numbers

The 16S rRNA and oligoalginate lyase gene of strain Kz7 have been deposited in GenBank under the accession numbers KF673475 and KF312482, respectively.

Results

Isolation and identification of strain Kz7

Through sole carbon source culturing, an alginate lyase-producing bacterium, Kz7, was isolated from Jiaozhou Bay, Qingdao, China. According to its morphological, physiological, and biochemical characteristics (data not shown) and the phylogenetic position (Supplementary Fig. S2) of its 16S rRNA gene sequence (KF673475), Kz7 was assigned to genus Shewanella and named Shewanella sp. Kz7.

Cloning and analysis of oligoalginate lyase gene

An oligoalginate lyase-encoding gene, oalS6, was isolated from Shewanella sp. Kz7. It consisted of an open reading frame of 2,313 bp in length. The deduced protein, OalS6, was composed of 770 amino acid residues, including a putative signal peptide (Met¹-Ala³⁹) with a calculated Mw of 85.2 kDa. In the previous study, we have cloned a PL family 17 oligoalginate lyase-encoding gene, oalS17 (GenBank accession number KJ094505), from the same strain (16). Interestingly, there were only 2-bp intervals between these two genes. These results indicated that oalS6 and oalS17 may be consecutively located in the same operon. However, OalS6 and OalS17 showed no significant sequence similarity with each other.

OalS6 had the highest amino acid identity (47%) with poly (beta-d-mannuronate) lyase AlyP (BAA01182.1) from Pseudomonas sp. OS-ALG-9 and 46% identity to a poly MG-specific alginate lyase AlyMG (AFC88009.1) from Stenotrophomonas maltophilia (25, 26). Both AlyP and AlyMG are endo-type alginate lyases and have been assigned as members of the PL-6 family (25, 26). As shown in the multiple sequence alignment, OalS6 had the same catalytic active and substrate-interacting sites conserved in the alginate lyases of the PL family 6 (Fig. 1). These results indicated that OalS6 was a new member of the PL family 6. Moreover, OalS6 has no significant sequence similarity with all known oligoalginate lyases. A phylogenetic tree was constructed for other known oligoalginate lyases and PL family 6 alginate lyases. The oligoalginate lyase OalS6, along with the other PL family 6 alginate lyases, AlyP and AlyMG, formed a deeply branched cluster in the phylogenetic tree (Fig. 2).

Fig. 1.

Protein sequence alignment of OalS6, chondroitin B lyase CslB (1dbg.pdb), polyMG specific alginate lyase AlyMG from Stenotrophomas maltophilia KJ-2, polyM-specific alginate lyase AlyP from Pseudomonas sp. OS-ALG-9 and putative alginate lyase Sfri-3103 from Shewanella frigidimarina NCIMB 400 using ClustalX. Identical amino acid residues are boxed in a dark shade, and amino acid residues above a 70% consensus are boxed in a pale shade. The filled triangle and filled circle indicate the proposed catalytic site and substrate-interacting site for chondroitin B lyase, respectively.

Fig. 2.

Phylogenetic analysis of oligoalginate lyase OalS6. The protein sequences of reported oligoalginate lyases and two PL-6 family endo-type alginate lyases (marked with an asterisk) were aligned using ClustalX, and phylogenetic tree was constructed using MEGA 4.0 via the neighbor-joining method.

OalS6 possessed a chondroitinase-like domain (Val⁴⁴ to Glu⁴¹⁹), which is conserved in the alginate lyases of PL-6 family; however, the oligoalginate lyases of the PL-15 family contain a heparinase II/III-like domain at the C-terminus, the oligoalginate lyases of the PL-17 family contain an alginate lyase superfamily domain at the N-terminus and a heparinase II/III family domain at the C-terminus, and the oligoalginate lyase AlyA5 in the PL-7 family contains an alginate lyase superfamily 2 domain (Supplementary Fig. S3). These findings indicated that OalS6 was structurally different from the other known oligoalginate lyases.

Expression and purification of OalS6

The oalS6 gene was over-expressed in the pET28a (+)/E. coli BL21(DE3) system. Most of the recombinant proteins existed in the soluble state when the cells were induced at 25°C for 6 h. The recombinant OalS6 protein was purified to homogeneity with a final yield of 71% using Ni-Sepharose column chromatography. Approximate 100 mg/l recombinant OalS6 was obtained via over-expression. The specific activity of the purified OalS6 towards alginate was 33.7 U/mg, which was much higher than other characterized oligoalginate lyases (10–17). The Mw of the recombinant OalS6 was estimated to be ∼85 kDa by SDS–PAGE (Fig. 3), which was in good agreement with the theoretical molecular mass.

Fig. 3.

SDS–PAGE of recombinant OalS6. Escherichia coli BL21(DE3) cells containing pET28a-oalS6 were cultured at 25°C for 6 h with or without 0.5 mM isopropyl-β-thiogalactoside (IPTG). M, protein markers; 1, BL21 cells containing recombinant plasmids without IPTG; 2, culture induced with 0.5 mM IPTG; 3, protein purified with His-tag affinity chromatography.

Biochemical characterization of OalS6

The optimal temperature of OalS6 was 40°C (Fig. 4a). When assayed in various pH values, the maximum activity of OalS6 was observed at pH 7.2 (Fig. 4b). More than 80% of the enzymatic activity was remained after being incubated below 40°C for 1 h (Fig. 4c). OalS6 retained over 80% of initial activity after incubated at pH 6.0–8.0 for 24 h (Fig. 4d).

Fig. 4.

Effects of acidity and temperature on the activity and stability of OalS6. (a) The optimal temperature of OalS6 determined by measuring the activity at various temperatures (10–60°C). (b) The optimal acidity of OalS6 determined by measuring the activity at 40°C in 50 mM Na2HPO4-citric acid (open triangle), 50 mM Na2HPO4-NaH2PO4 (open rhombus), 100 mM Tris-HCl (filled square) and 50 mM Gly-NaOH (filled circle). (c) The thermostability of OalS6 studied by measuring the residual activity after incubation at different temperatures and in 20 mM phosphate buffer (pH 7.0) for 1 h. (d) Acidity stability of OalS6 determined by measuring the residual activity at 40°C in 20 mM phosphate buffer (pH 7.0) after incubation in the buffers stated above at 4°C for 6 h.

OalS6 did not require NaCl for its enzymatic activity; however, its activity was significantly enhanced by NaCl (Fig. 5a). The specific activity of OalS6 was increased by 4.76 times in the presence of a low salinity (50 mM NaCl). The activity of OalS6 was also enhanced by K⁺. When determined the effect of di- and trivalent metal ions (1 mM) on the activity of OalS6, Cu²⁺, Zn²⁺, Ni²⁺, Al³⁺ and Fe³⁺ obviously inhibited the activity of OalS6 (P < 0.01), while Ba²⁺, Mn²⁺ and Ca²⁺ was not significant effective on its activity (P > 0.05). The chelating agent, EDTA (1 mM), and surfactant agent, SDS (1 mM), also obviously inhibited the activity of OalS6 (P < 0.01) (Fig. 5b).

Fig. 5.

Effects of reagents on the activity of OalS6. (a), Effects of different concentrations of NaCl and KCl on the activity of OalS6. (b), Effects of 1 mM metal ions, EDTA and SDS on the activity of OalS6. Activity without addition of metal ions or chemical agents was defined as 100%. Data were shown as means ± SD (n = 3).This is a typical result of three experiments. * represents P < 0.05; ** represents P < 0.01.

Substrate specificity and kinetic parameters of OalS6

The substrate specificity of OalS6 was determined using alginate, polyG, polyM and chondroitin sulphate as substrates. Among the tested polymeric substrates, OalS6 was most active on polyG block (79.4 ± 9.2 U/mg) compared to polyM block (3.2 ± 0.6 U/mg) and native alginate (33.7 ± 4.4 U/mg) (Table I). These results were further confirmed by TLC chromatography (Supplementary Fig. S4). In addition, OalS6 showed no activity on chondroitin sulphate.

Table I.

Kinetic parameters of OalS6 for different substrates

Substrates	Specific activity (U/mg)	Km (mg/ml)	Vmax (U/mg)	kcat/Km (U mg−1 s−1)
Alginate	33.7 ± 4.4	0.91 ± 0.04	68.8 ± 12.5	61.7 ± 3.1
PolyG	79.4 ± 9.2	0.37 ± 0.07	121.1 ± 7.2	102.3 ± 1.1
PolyM	3.2 ± 0.6	6.71 ± 0.50	2.9 ± 0.2	2.4 ± 0.1

The kinetic parameters of OalS6 towards alginate, polyM and polyG were measured at various substrate concentrations and calculated according to the Lineweaver–Burk formula (Supplementary Fig. S5). The Km, Vmax and kcat/Km of OalS6 for the cleavage of various alginate polymers are shown in Table II. The apparent Km of OalS6 against alginate, polyG, polyM was 0.91 ± 0.04 mg/ml, 0.37 ± 0.07 mg/ml and 6.71 ± 0.50 mg/ml, respectively. The catalytic efficiency values (kcat/Km) of OalS6 against alginate, polyG and polyM were 61.7 ± 3.1 mg/ml·s, 102.3 ± 1.1 mg/ml·s and 2.4 ± 0.1 mg/ml·s, respectively. The specific activity and kinetic parameters of OalS6 showed that it was a polyG preferred enzyme. In the previous study, a polyM preferred oligoalginate lyase, OalS17, was cloned and characterized from the same strain. Combination of OalS6 (0.5 U) and OalS17 (0.5 U) showed an activity of 1.41 U towards alginate. The degradation products of OalS6 alone, OalS17 alone and OalS6 plus OalS17 at different reaction times (0, 1, 5 and 10 min) were shown in Supplementary Fig. S6.

Table II.

¹H (600 MHz) and ¹³C NMR (150 MHz) data for compounds 1a and 1b in D₂O

Position	1a		1b
	δC	δH (J in Hz)	δC	δH (J in Hz)
2	107.2, C		106.5, C
3	46.6, CH2	2.07, dd (14.3, 2.6)	46.1, CH2	2.29, dd (13.7, 6.0)
		2.55, dd (14.3, 7.1)		2.55, dd (13.7, 7.0)
4	74.3, CH	4.45, m	74.5, CH	4.48, m
5	91.6, CH	4.14, dd (3.4, 3.8)	91.0, CH	3.88, dd (4.4, 6.2)
6	92.2, CH	5.02, d (3.8)	92.9, CH	5.00, d (6.2)
2-COOH	179.7, C		179.2, C

Action model and reaction products of OalS6

The mode of action of OalS6 was monitored by size-exclusion chromatography using polyG block as the substrate (Fig. 6). Monomeric sugar acid was detected at a reaction time of 1 min, and intermediates were not found during an entire 30 min reaction. These results indicate that OalS6 is an exo-type alginate lyase.

Fig. 6.

FPLC analysis of the degradation products of polyG block by OalS6. The polyG block (5 mg/ml) was degraded by OalS6 (0.6 mg/ml) at 40°C. The vertical dotted line in the chromatogram indicates the injection time. The elution volume of unsaturated monomer is 17.5 ml.

As showed in Fig. 7, after alginate disaccharides (10 mg/ml) were incubated with OalS6 (0.1 mg/ml) for 60 min, the alginate disaccharides were completely degraded into monosaccharides. These results indicated that OalS6 showed a high affinity for alginate disaccharides and efficiently depolymerized alginate disaccharides into monosaccharides.

Fig. 7.

Analyses of the degradation products of disaccharide by OalS6. (a) TLC analysis of the reaction products from disaccharide. The reactions were performed with OalS6 at 40°C in 20 mM phosphate buffer (pH 7.0). M, The purified monomeric sugar, dimer and trimer standards. (b–d) FPLC analysis of the reaction products from disaccharide at various times (0, 30, 60 min). The elution volumes of unsaturated monomer and dimer are 17.5 and 16.4 ml, respectively.

The main OalS6-degrading products of polyG block were determined with ESI-MS, 1D ¹H-NMR, ¹³C-NMR and 2D ¹H-¹H COSY NMR spectra. As shown in the ESI-MS analysis (Supplementary Fig. S7), a molecular ion peak appeared at 193.0359 m/z [M-H]⁻. The ¹H-NMR and DEPTQ data were shown in Table II. It identified the presence of one methylene (δ_C/H 46.6/2.07, 2.55), two oxygenated methines, one double oxygenated methine, one double oxygenated quarternary carbon and one carboxylic carbon (δ_C 179.7) (Table II). ¹H-¹H COSY correlations from H₂-3 to H-4 (δ_H4.45) and H-5 (δ_H 4.14) and finally to H-6 (δ_H 5.02) revealed the existence of a CH₂ (3)-CH (4)-CH (5)-CH (6) moiety (Supplementary Fig. S8). In association with the presence of the double oxygenated methine (δ_C/H 92.2/5.02) and quarternary carbon (δ_C 107.2), these findings indicate that the main product was 2, 4, 5, 6-tetrahydroxytetrahydro-2H-pyran-2-carboxylic acid (TPC) (1a and 1b, Supplementary Fig. S9), which were two cyclic hemiacetal stereoisomers formed by predominantly hydration of the reported DEH molecules. The absence of the ketone and aldehyde signals in the NMR data definitely indicated that the main product was not DEH.

Discussion

Alginate, the main component of brown macroalgae, is utilized as biomass by various marine bacteria (27). Bacteria synthesize various types of enzymes to metabolize alginate. For example, endo-type alginate lyases depolymerize alginate into oligosaccharides, and exo-type oligoalginate lyases further degrade oligosaccharides into monosaccharides (5, 11). The majority of alginate lyases reported to date are endo-type enzymes, while there are only a few exo-type oligoalginate lyases belonging to the PL family 7, 14, 15 and 17 (10–17).

In this study, the first exo-oligoalginate lyase in the PL-6 family, OalS6, was identified. OalS6 does not have significant sequence similarity with other known oligoalginate lyases. It contains a chondroitinase-like domain at the N-terminus, different from other oligoalginate lyases (Supplementary Fig. S3). These findings indicate that OalS6 may structurally different from other known oligoalginate lyases. Notably, although other alginate lyases in the PL-6 family also possess the chondroitinase-like domain, they are endo-type lyases, yielding unsaturated di- and tri-saccharides as their main products (25, 26).

The oligoalginate lyases reported to date were predominantly polyM block preferred enzymes. Oligoalginate lyases in PL-15 and 17 family exhibited high activity towards polyM blocks (11, 12, 15–17), whereas only one oligoalginate lyase (AlyA5) in the PL-7 family was much more active on polyG block (14). However, the application of AlyA5 was limited by narrow range of pH and temperature tolerance (14). In this study, OalS6 was a polyG block preferred oligoalginate lyase (Table II) and it revealed a better thermostability and pH stability (Fig. 4). In addition, OalS6 appeared to have the highest specific activity (33.7 ± 4.4 U/mg) towards alginate among all characterized oligoalginate lyases, such as OalA, OalB and OalC from V. splendidus 12B01 (28.5 U/mg, 20 U/mg and 20.8 U/mg, respectively); A1-IV (16.1 U/mg) from Sphingomonas sp. A1 and Aly17C (1.98 U/mg) from S. degradans 2–40 (10–15, 17). Recently, a polyM block preferred oligoalginate lyase OalS17 (32 U/mg) from Shewanella sp. Kz7 has been cloned and characterized in our lab (16). Combination of OalS6 and OalS17 showed an obviously synergistic alginate-degrading effect. Thus, it is an intelligent strategy that the polysaccharide degrading organisms expressed two oligoalginate lyases with complementary substrate specificity to obtain efficient alginate degrading effect.

As documented previously, oligoalginate lyases degrade both alginate oligomeric and alginate polymer mainly into unsaturated monosaccharides. Then the unsaturated monosaccharide is non-enzymatically converted to DEH (28). Recently, Enquist-Newman et al.. (4) showed that DEH molecules in the oligoalginate lyases-degrading products of alginate were predominantly hydrated to form two cyclic hemiacetal stereoisomers; in addition, these hydrated DEH molecules could still fed into ED pathway to produce ethanol. We also confirmed that the main OalS6-degrading products of polyG block were TPC (Supplementary Fig. S9).

In conclusion, OalS6 is the first exo-type oligoalginate lyase belonging to the PL-6 family. It preferred to degrade polyG block into monosaccharides, which then transformed into TPC automatically. OalS6 is useful for producing monomeric sugar acid from brown macroalgae and further developing biofuel.

Supplementary Data

Supplementary Data are available at JB Online.

Glossary

Abbreviations

CAZY

carbohydrate-active enzymes

DEH

4-deoxy-l-erythro-5- hexoseulose uronic acid

ED

Entner–doudoroff

KDG

2-keto-3-deoxygluconate

Mw

molecular weight

PL family

polysaccharide lyase family

SDS–PAGE

sodium dodecyl sulphate polyacrylamide gel electrophoresis

TPC

2, 4, 5, 6-tetrahydroxy-pentahydro-pyran-2-carboxylic acid

Funding

This work was supported by the NSFC-Shandong Joint Fund for Marine Science Research Centers (U1406402) and National High-tech R&D Program of China (2014AA093504), National Natural Science Foundation of China (31000361 and 41376144) and Special Fund for Marine Scientific Research in the Public Interest (201105027-3 and 201005024), Science and Technology Development Project of Shandong Province (2014GGH215002).

Conflict of Interest

None declared.