Elucidation of degrading pattern and substrate recognition of a novel bifunctional alginate lyase from Flammeovirga sp. NJ-04 and its use for preparation alginate oligosaccharides
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The alginate oligosaccharides have been widely used in agriculture, medicine, and food industries due to their versatile physiological functions such as antioxidant, anticoagulant, and antineoplastic activities. The bifunctional alginate lyases can degrade the alginate polysaccharide more efficiently into alginate oligosaccharides. Therefore, it is crucial to discover new bifunctional alginate lyase for alginate oligosaccharide production.
Herein, a novel bifunctional alginate lyase FsAlgB was cloned and identified from deep-sea bacterium Flammeovirga sp. NJ-04, which exhibited broad substrate specificity and the highest activity (1760.8 U/mg) at pH 8.0 and 40 °C. Furthermore, the Km values of FsAlgB towards polyG (0.69 mM) and polyMG (0.92 mM) were lower than that towards sodium alginate (1.28 mM) and polyM (2.06 mM). Recombinant FsAlgB was further characterized as an endolytic alginate lyase, and it can recognize the tetrasaccharide as the minimal substrate and cleave the glycosidic bonds between the subsites of − 3 and + 1.
This study provided extended insights into the substrate recognition and degrading pattern of alginate lyases with broad substrate specificity.
KeywordsAlginate lyase Bifunctional Oligosaccharide Action pattern Substrate recognition
degree of depolymerization
sodium dodecyl sulfate polyacrylamide gel electrophoresis
Alginate is the main component of the cell wall of brown algae such as Laminaria japonica . It is a linear acidic polysaccharide consisting of β-d-mannuronate (M) and its C5 epimer α-l-guluronate (G). These units are arranged into three different kinds of blocks, namely poly β-d-mannuronate (polyM), poly α-l-guluronate (polyG) and the heteropolymer (polyMG) . As the most abundant marine biomass and low-cost material, alginate has been widely used in food and medical industries due to its favorable chemical properties and versatile activities. Insufficiently, the applications of this linear polysaccharide have been greatly limited due to its high molecular weight and low bioavailability. Recently, the degradation products of alginate, namely the alginate oligosaccharides, have attracted increasing attention due to their biological activities and excellent solubility. For instance, they have been widely used as plant growth accelerators, anticoagulants, and tumor inhibitors due to their versatile physiological functions such as antioxidant, anticoagulant, and antineoplastic activities [3, 4, 5]. Moreover, they can also regulate the blood sugar and lipid content . As a result, it is more promising to prepare the functional oligosaccharides by utilizing the abundant polysaccharides.
Alginate lyase and can degrade alginate into unsaturated oligosaccharides by β-elimination . On the basis of their substrate specificities, alginate lyase can be classified into G block-specific lyase (polyG lyase, EC220.127.116.11) and M block-specific lyase (polyM lyase, EC18.104.22.168) . While according to the mode of action, it can be sorted into endolytic and exolytic ones . Endolytic enzymes can recognize and cleave glycosidic bonds inside alginate polymers with unsaturated oligosaccharides (such as di-, tri-, and tetra-saccharides) as main products , while exolytic ones can further degrade oligosaccharides into monomers . In addition, according to protein sequence similarity, alginate lyases are organized into the PL-5, 6, 7, 14, 15, 17, and 18 families (http://www.cazy.org/fam/acc_PL.html) .
So far, alginate lyases have been widely used to elucidating fine structures of the alginate and prepare protoplast [13, 14]. Moreover, it also shows great potential in treating cystic fibrosis by degrading the polysaccharide biofilm of pathogen bacterium such as Pseudomonas aeruginosa [15, 16].
In this study, we cloned and identified a novel bifunctional alginate lyase of PL7 family FsAlgB. Like most of alginate lyases of PL 7 family, it can degrade alginate into oligosaccharides in an endolytic manner . It is reported that the A1-II′ from Sphingomonas sp. A1, a member of PL 7 family, displays a β-sandwich jelly roll-fold, and can bind tetrasaccharide as its minimal substrate and hydrolyzed it into disaccharide and trisaccharide. The structure of A1-II′ complexed with tetrasaccharide indicated that Gln189 and Arg146 act as a neutralizer for the substrate carboxyl group, His191 as a general base, and Tyr284 as a general acid . However, the substrate degradation mode of PL 7 family enzymes is still unclear. The Flammeovirga sp. NJ-04 is an alginate-degrading bacterium isolated from deep-sea area and a gene cluster for degrading alginate has been identified . In this paper, the degradation mode of FsAlgB was discussed, and the biochemical characteristics, action mode, and product analysis were also studied.
Materials and strains
Sodium alginate from Macrosystis pyrifera (M/G ratio 77/23) was purchased from Sigma-Aldrich (viscosity ≥ 2000 Cp, St. Louis, MO, USA). PolyM and polyG (purity: about 95%; M/G ratio: 97/3 and 3/97; average degree of depolymerization (DP): 39; average molecular weight: 7200 Da) were purchased from Qingdao BZ Oligo Biotech Co., Ltd (Qingdao, China). PolyMG (M/G ratio 48/52, average DP: 50, and average molecular weight: 8000 Da) was donated by the bioengineering group of our college. Marine bacterium Flammeovirga sp. NJ-04 was previously isolated from South China Sea and conserved in our laboratory . Escherichia coli DH5α was used for plasmid construction, and E. coli BL21 (DE3) was used for gene expression. These two strains were grown in Luria–Bertani (LB) broth or on LB broth agar plates (LB broth supplemented with 1.5% agar) containing 100 μg/mL ampicillin.
The conserved domains of FsAlgB were predicted using the InterProScan 4 running the HMM Pfam application (http://www.ebi.ac.uk/Tools/pfa/iprscan/). The homology protein sequence was performed with NTI vector. The phylogenetic tree was constructed based on related alginate lyase protein sequences of PL 7 family using Molecular Evolutionary Genetics Analysis (MEGA) Program version 6.0. The homology modeling and docking was built by Protein Homology/analogY Recognition Engine V 2.0.
Expression and purification of FsAlgB
As previously reported, the strain Flammeovirga sp. NJ-04 was identified to be close to Flammeovirga sp. OC4 . Therefore, the primers for cloning FsAlgB were designed on the basis of sequence of putative alginate lyase gene sequence (WP_044204802.1) within the genome of Flammeovirga sp. OC4. The FsAlgB gene was amplified with primers designed as follows: the forward primer: 5′-GGCCATATGATGAACAGACTTTTTACTTT-3′ and the reverse primer: 5′-GGCCTCGAGTTGATGTGTTACCGACAAGT-3′ from the genomic DNA of Flammeovirga sp. NJ-04.
The alginate lyase gene was subcloned and ligated into pET-21a (+) expression vector. The recombinant E. coli BL21 (DE3) harboring the pET-21a (+)/FsAlgB was cultured in an LB medium (containing 100 μg ampicillin/mL) for 2–3 h with shaking at 200 rpm and 37 °C up to an OD600 of 0.4–0.6. The cells were induced by adding 0.1 mM IPTG and then cultured at 20 °C for 30 h. The purification of FsAlgB was carried out as previously described as follows . The cell homogenate containing the recombinant protein was loaded onto Ni–NTA Sepharose column (GE Healthcare, Uppsala, Sweden) equilibrated with lysis buffer. The column was washed with wash buffer [50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 20 mM imidazole], and the recombinant enzyme was eluted with elution buffer [50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 300 mM imidazole]. The active fraction was collected and desalted using HiTrap™ desalting column (Amersham Biosciences, Buckinghamshire, UK) and then analyzed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Substrate specificity and enzymatic kinetics
The activity of FsAlgB was determined according to the ultraviolet absorption method described by Inoue . The reaction system was constructed as follows: the purified enzyme (0.1 mL 50 mM Tris-HCl buffer of pH 8.0 containing 0.042 mg of enzyme) was mixed with 0.9 mL substrate in 50 mM Tris-HCl buffer with 1% sodium alginate (pH 8.0) and the mixture was incubated at 40 °C for 10 min. The reaction was stopped by heating in boiling water for 10 min. The enzyme activity was then determined by measuring the increased absorbance at 235 nm. One unit was defined as the amount of enzyme required to increase the absorbance at 235 nm by 0.1 per min. Each experiment consisted of three replicates. In addition, the protein concentrations were determined by a protein quantitative analysis kit (Beyotime Institute of Biotechnology, Nantong, China).
To investigate the substrate specificity, the purified FsAlgB (~ 0.19 ng) in 1 mL of 50 mM Tris-HCl buffer of pH 8.0 was reacted with 1% of sodium alginate, polyMG, polyM, and polyG, respectively. The assay of enzyme activity was defined as described previously. The kinetic parameters of the FsAlgB towards these four kinds of substrates were determined by measuring the enzyme activity with substrates at different concentrations (0.1–8.0 mg/mL). The concentrations of the substrates and velocity (V) were calculated as previously reported . The concentrations of the product were determined by monitoring the increase in absorbance at 235 nm using the extinction coefficient of 6150 M−1 cm−1. Velocity (V) at the tested substrate concentration was calculated as follows: V (mol/s) = (milliAU/min × min/60 s × AU/1000 milliAU × 1 cm)/(6150 M−1 cm−1) × (2 × 10−4 L). The Km and Vmax values were calculated by hyperbolic regression analysis as previously described [22, 23]. The turnover number (kcat) of the enzyme was calculated by the ratio of Vmax versus enzyme concentration ([E]). Each experiment consisted of three replicates.
Biochemical characterization of FsAlgB
The effects of temperatures (20–80 °C) on the purified FsAlgB in 50 mM Tris-HCl buffer of pH 8.0 were investigated at pH 8.0. The thermal stability of the enzyme was determined under the standard assay conditions after incubating the purified FsAlgB at 20–80 °C for 30 min. In addition, the thermally induced denaturation was also investigated by measuring the residual activity after incubating the enzyme at 20–40 °C for 0–180 min. The effects of pH on the enzyme activity of FsAlgB were evaluated by incubating the purified enzyme in buffers with different pH (4.0–11.0) under the standard conditions. The buffers with different pHs used are 50 mM phosphate citrate (pH 4.0–5.0), 50 mM NaH2PO4-Na2HPO4 (pH 6.0–8.0), 50 mM Tris-HCl (pH 7.0–9.0), and glycine-NaOH (pH 9.0–10.0). The pH stability was characterized by determining the residual activity after the enzyme was incubated in buffers with different pH (4.0–11.0) for 24 h. Each experiment consisted of three replicates.
The influences of metal ions on the activity of FsAlgB were performed by incubating the purified enzyme in 50 mM Tris-HCl buffer of pH 8.0 at 4 °C for 24 h in the presence of various metal compounds with a final concentration of 1 mM. Then, the activity was measured under standard conditions and the reaction mixture without any metal ion was taken as 100%. The effects of NaCl on enzyme activity of FsAlgB were measured in buffers with different concentrations of NaCl (0–800 mM) and the maximal enzyme activity served as control. Each experiment consisted of three replicates.
Action pattern and degradation product analysis
To determine the smallest substrate and identify the number of substrate-binding subsites of FsAlgB, hydrolysis reactions (10 μL reaction mixture) were performed at 40 °C for 24 h using FsAlgB in 50 mM Tris-HCl buffer of pH 8.0 and oligosaccharides (10 mg/mL) with different DPs (DP2–8). The degradation products were analyzed by ESI-MS in a positive-ion mode using the following settings: ion source voltage, 4.5 kV; capillary temperature, 275–300 °C; Tube lens, 250 V; sheath gas, 30 arbitrary units (AU); scanning mass range, 150–2000 m/z.
To determine the cleavage selectivity of the enzyme, the ESI-MS was used to analyze the degradation products in the hydrolysis procedure of FsAlgB toward sodium alginate, polyMG, polyM, and polyG, respectively. The reaction mixtures (800 μL containing 1 μg purified enzyme and 2 mg substrates) were incubated at 40 °C for 72 h. Then, the mixtures were centrifuged at 8000g rpm for 10 min to remove the undissolved materials. In addition, the hydrolysates were loaded onto a carbograph column (Alltech, Grace Davison Discovery Sciences, United Kingdom) to remove salts, and then, the eluate was concentrated, dried and re-dissolved in 1 mL methanol. The degradation products were then analyzed by TLC plate as previously reported . In brief, the degradation products were developed by TLC plate (TLC silica gel 60 F254, Merck KGaA, Darmstadt, Germany) with the solvent system (1-butanol/acetic acid/water 3:2:3) and visualized by heating TLC plate at 130 °C for 5 min after spraying with 10% (v/v) sulfuric acid in ethanol. The ESI-MS was also employed to determine the composition of the hydrolysates using analysis conditions as described above.
Molecular modeling and docking analysis
The three-dimensional structure of FsAlgB was constructed using Protein Homology/analogY Recognition Engine V 2.0 on the basis of homologues of known structure (alginate lyase A1-II′ from Sphingomonas sp. A1 with PDB ID: 2ZAB) with sequence similarity of 39%. The molecular docking of the FsAlgB and tetrasaccharide (GGGG) was performed using MOE (Molecular Operating Environment, Chemical Computing Group Inc., Montreal, Canada). The ligand-binding sites were defined using the bound ligand in the crystal structures. PyMOL (http://www.pymol.org) was used to visualize and analyze the modeled structure and to construct graphical presentations and illustrative figures.
Sequence analysis of the alginate lyase gene
The gene FsAlgB was cloned and sequenced (accession number: MG063276). As shown in Additional file 1: Figure S1, the open reading frame (ORF) of FsAlgB consists of 900 bp and encodes a putative alginate lyase composed of 299 amino acids with a theoretical molecular mass of 34.50 kDa. According to the conserved domain analysis, FsAlgB possesses only a C-terminal catalytic domain consisting of 231 amino acids (Thr54–Glu284).
Cloning and expression of FsAlgB
Substrate specificity and kinetics of FsAlgB (data shown are the mean ± SD, n = 3)
1760.8 ± 15.8
2103.2 ± 21.3
1124.9 ± 8.2
2445.6 ± 12.7
1.28 ± 0.28
0.92 ± 0.04
2.06 ± 0.17
0.69 ± 0.11
0.12 ± 0.03
0.15 ± 0.01
0.08 ± 0.01
0.18 ± 0.04
2.22 ± 0.19
2.73 ± 0.23
1.48 ± 0.07
3.33 ± 0.21
1.73 ± 0.32
2.97 ± 0.41
0.72 ± 0.17
4.83 ± 0.39
The kinetics of FsAlgB towards sodium alginate, polyMG, polyM, and polyG were calculated according to the hyperbolic regression analysis. The non-linear fit curves for the degradation of the four kinds of substrates are shown in Additional file 1: Figure S2. As shown in Table 1, the Km values of FsAlgB with sodium alginate, polyMG, polyM, and polyG as substrates were 1.28 mM, 0.92 mM, 2.06 mM, and 0.69 mM, respectively. Thus, FsAlgB had a much lower Km values towards polyG and polyMG, indicating that it exhibited higher affinity towards G block than that to M block. The kcat values of FsAlgB towards sodium alginate, polyMG, polyM, and polyG were 2.22 s−1, 2.73 s−1, 1.48 s−1, and 3.33 s−1, respectively. It indicated that FsAlgB exhibited the higher catalytic efficiency towards G block and hybrid MG block than that towards M block.
Biochemical characterization of FsAlgB
Effects of metal ions and chemical agents on FsAlgB activity (data shown are the mean ± SD, n = 3)
Relative activity (%)
100 ± 2.1
127.2 ± 2. 9
104.2 ± 3.2
109.2 ± 3.3
57.2 ± 2.0
97.3 ± 2.5
95.4 ± 3.4
79.3 ± 4.8
46.7 ± 2.5
Action pattern analysis and substrate docking of FsAlgB
TLC and ESI-MS analysis of degradation products
As described in our previous work, Flammeovirga sp. NJ-04 was previously isolated from South China Sea and exhibited versatile abilities for degrading algal polysaccharides. In addition, an endolytic alginate lyase, FsAlgA, with heat stability has been cloned and identified from the strain . In this study, a new endo-type alginate lyase was identified from Flammeovirga sp. NJ-04 and its action pattern was further elucidated. The sequence alignment indicated that FsAlgB shares the highest identity of 65% with FsAlgA, which indicated that it is a new member of PL 7 family. In addition, it contained three conserved regions, namely “PRT/V/SELRE”, “YFKA/VGN/VY”, and “QIH”. Especially, the region of QIH was reported to be involved in substrate specificity; the residue “I” may recognize the polyG block or MG blocks . Thus, it was reasonable that FsAlgB preferred polyG as the substrate just as other alginate lyases containing the QIH region, such as ALY-1 from Corynebacterium sp. strain ALY-1 , A1-I from Sphingomonas sp. A1 , and A1m from Agarivorans sp. JAM-A1m , while, for other alginate lyases containing the QVH region of PL 7 family, polyM is the optimal substrate, such as alginate lyaseA9m from Vibrio sp. JAM-A9m , alginate lyases AlyVOA, and AlyVOB from Vibrio sp. O2, degraded in activity assays .
While compared with the other PL 7 family enzymes, FsAlgB had lower Km values. For instance, AlgMsp from Microbulbifer sp. 6532A possessed Km values of 1.8–6.8 mM towards different substrates . AlyA1 from Zobellia galactanivorans DsiJT showed Km values varying from 1.7 mM to 6.2 mM . The kcat/Km values of FsAlgB towards polyG (4.83 mM−1 s−1) and polyMG (2.97 mM−1 s−1) were higher than to sodium alginate (1.73 mM−1 s−1) and polyM (0.72 mM−1 s−1), which indicated that the enzyme showed higher catalytic efficiency towards polyG and polyMG than to polyM.
As to biochemical characteristics, most of alginate lyases exhibit the optimal activity in the range of pH 7–8.5. For instance, FsAlgA from Flammeovirga sp. NJ-04 , AlyA1 from Zobellia galactanivorans , and Alg7D from Saccharophagus degradans  show the optimal activity at pH 7.0, while AlgMsp from Microbulbifer sp. 6532A , Algb from Vibrio sp. W13 , and A1-II′ from Sphingomonas sp. A1  possess the optimal pH of 8.0. As to the optimal temperature, most of alginate lyases exhibit the optimal activity around 30–40 °C, while FlAlyA from Flavobacterium sp. UMI-01 had a highest optimal temperature of 55 °C . In addition, AlgMsp from Microbulbifer sp. 6532A  and Alg7D from Saccharophagus degradans  displayed their maximal activity at 50 °C. Moreover, they could maintain most of the activities below 40 °C, except the Alg7D from Saccharophagus degradans which showed lower heat stability and retained only 40% of its maximum activity below 30 °C.
The effects of metal ions on FsAlgB were similar to that on Aly510-64 from Vibrio sp. 510-64  and AlyAL-28 from Vibrio harveyi AL-28 . Specifically, the activity of FsAlgB can be enhanced by NaCl with different concentrations (100–600 mM) and the maximal activity can be reached with 300 mM NaCl (Fig. 4d). Therefore, FsAlgB is a salt-activated alginate lyase. Since these salt-activated enzymes are usually isolated from marine bacteria, it is easy to understand the activation of NaCl . Furthermore, it is assumed that high ionic strength might be essential to maintain the uronic acid units at a minimal interunit period for proper fitting of the enzyme . The enhanced effects of NaCl with high concentration may be partly caused by the removal of bound water from alginate molecules or the stabilization of the transition state .
As to the action mode, ALY-1 from Corynebacterium sp. ALY-1 could degrade oligosaccharides larger than pentasaccharides, indicating that it possessed a subsite corresponding to hexasaccharide unites. The action pattern of the enzyme towards hexamers was confirmed by the HPLC analysis, and the main products were identified to be dimers and tetramers, which suggested that the catalytic site of the enzyme was matched to the linkage the second (− 2) and the third (− 1) residues from the nonreducing end . Aly272 from Alteromonas sp. No272 exhibited no activity towards trimeric mannuronate or guluronate, but can degrade oligosaccharides larger than tetramers. The kinetic analysis kcat/Km combined with intrinsic reaction rate constant (kint) and intrinsic substrate-binding constant (kint) indicated that it most likely consisted of six binding sites . Almost all alginate lyases of PL7 family are endolytic enzymes and can degrade the alginate into oligosaccharides with low DP of 2–5 as the main product. Exceptionally, AlyA5 from Zobellia galactanivorans can release disaccharides in an exolytic manner . Due to various biological activities, alginate oligosaccharides have been widely used in various fields [37, 38]. While considering the drawbacks and defects of chemical method, the alginate lyases, especially the enzymes with broad substrate specificity and high activity, are crucial in producing functional oligosaccharides. FsAlgB is a novel alginate lyase with broad substrate specificity and high activity, which can effectively degrade alginate and produce oligosaccharides with lower DPs, so it may be a potent tool to produce oligomers. Furthermore, this work also provided extended insights into the substrate recognition and degrading pattern of alginate lyases with broad substrate specificity.
In this study, a novel bifunctional alginate lyase FsAlgB has been identified from Flammeovirga sp. NJ-04, which exhibited the highest activity (1760.8 U/mg) at pH 8.0 and 40 °C. In addition, it possessed broad substrate specificity, showing high activities towards not only polyM (polyβ-d-mannuronate) but also polyG (poly α-l-guluronate). And for further applications in industry, its action mode was also elucidated. Remarkably, FsAlgB can recognize the tetrasaccharide as the minimal substrate and cleave the glycosidic bonds between the subsites of − 3 and + 1 within the polysaccharide producing oligosaccharides with DP of 2–4. TLC and ESI-MS analysis indicated that it can degrade the substrates in an endolytic manner to release a series of oligosaccharides. Thus, with high activity, broad substrate specificity, and product specificity, it might be a potential candidate for degrading alginate to produce oligosaccharides.
BWZ and LMN conducted experiments. All authors designed experiments and interpreted data and BWZ with FN drafted the manuscript, while funding was secured by YS and ZY. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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The work was supported by the National Natural Science Foundation of China (Grant Nos. 31601410, 81503463).
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