Synergy of the Two Alginate Lyase Domains of a Novel Alginate Lyase from Vibrio sp. NC2 in Alginate Degradation

ABSTRACT

Alginate lyases play a vital role in the degradation of alginate, an important marine carbon source. Alginate is a complex macromolecular substrate, and the synergy of alginate lyases is important for the alginate utilization by microbes and the application of alginate lyases in biotechnology. Although many studies have focused on the synergy between different alginate lyases, the synergy between two alginate lyase domains of one alginate lyase has not been reported. Here, we report the synergism between the two catalytic domains of a novel alginate lyase, AlyC6’, from the marine alginate-degrading bacterium Vibrio sp. NC2. AlyC6’ contains two PL7 catalytic domains (CD1 and CD2) that have no sequence similarity. While both CD1 and CD2 are endo-lyases with the highest activity at 30°C, pH 8.0, and 1.0 M NaCl, they also displayed some different properties. CD1 was PM-specific, but CD2 was PG-specific. Compared with CD2, CD1 had higher catalytic efficiency, but lower substrate affinity. In addition, CD1 had a smaller minimal substrate than CD2, and the products from CD2 could be further degraded by CD1. These distinctions between the two domains enable them to synergize intramolecularly in alginate degradation, resulting in efficient and complete degradation of various alginate substrates. The bioinformatics analysis revealed that diverse alginate lyases have multiple catalytic domains, which are widespread, especially abundant in Flavobacteriaceae and Alteromonadales, which may secret multimodular alginate lyases for alginate degradation. This study provides new insight into bacterial alginate lyases and alginate degradation and is helpful for designing multimodular enzymes for efficient alginate depolymerization.

IMPORTANCE Alginate is a major component in the cell walls of brown algae. Alginate degradation is carried out by alginate lyases. Until now, while most characterized alginate lyases contain one single catalytic domain, only a few have been shown to contain two catalytic domains. Furthermore, the synergy of alginate lyases has attracted increasing attention since it plays important roles in microbial alginate utilization and biotechnological applications. Although many studies have focused on the synergy between different alginate lyases, the synergy between two catalytic domains of one alginate lyase has not been reported. Here, a novel alginate lyase, AlyC6’, with two functional alginate lyase domains was biochemically characterized. Moreover, the synergism between the two domains of AlyC6’ was revealed. Additionally, the distribution of the alginate lyases with multiple alginate lyase domains was investigated based on the bioinformatics analysis. This study provides new insight into bacterial alginate lyases and alginate degradation.

INTRODUCTION

Brown algae are a type of marine biomass with a very high output (1). Alginates are linear unbranched polysaccharides existing in great abundance in the cell walls of brown algae, accounting for approximately 30 to 60% dry weight of brown algae (1). Therefore, alginates act as a substantial fraction of marine biomass and are an important source of marine carbon cycling (2). Alginate consists of two residues, β-d-mannuronate (M) and its C5 epimer, α-l-guluronate (G) (3). These two residues are linked homogeneously or heterogeneously by 1→4 glycosidic bonds, which contribute to three different blocks, polymanuronate (PM) blocks, polyguluronate (PG) blocks, and polyMG/GM (PMG) blocks (4, 5). Alginate has wide applications as a viscosity-enhancing hydrocolloid, gelling agent, and food and cosmetics additive (6 to 9).

Alginate lyases are synthesized by marine algae, marine mollusks, fungi, bacteria, and viruses, which play important roles in the degradation and assimilation of alginate (10, 11). Alginate lyase degrades alginate via a β-elimination reaction, producing unsaturated bonds at the nonreducing end of one of the cleavage products (12, 13). Based on the amino acid sequence, alginate lyases are distributed into the polysaccharide lyase (PL) 5, 6, 7, 8, 14, 15, 17, 18, 31, 32, 34, 36, 39, and 41 families in the carbohydrate-active enZYmes database (http://www.cazy.org/) (14 to 17). Alginate lyases are classified into three groups by their substrate specificity: PG-specific lyases (EC 4.2.2.11), PM-specific lyases (EC 4.2.2.3), and bifunctional lyases that can degrade both PM and PG (EC 4.2.2.-). According to the action mode, alginate lyases are grouped into endotype lyases that generate oligosaccharides by cleaving the inside-chain glycosidic bonds and exotype lyases producing monomers or dimers by gradual degradation from the end of alginate (1, 18, 19). Alginate lyases play an important role in marine alginate decomposition and recycling. They also play a remarkable role in biotechnological applications (20). Particularly, alginate lyases are essential for unsaturated alginate oligosaccharides production and have potentials in biofuel and other biochemical preparations (1, 21).

Until now, while most characterized alginate lyases in the CAZy database contain one single catalytic domain, only a few have been shown to contain two catalytic domains. Algb from Vibrio sp. W13 contains two alginate lyase domains, the activities of which, however, have not been verified (22). AlyA from Vibrio splendidus 12B01 also has two annotated alginate lyase domains, but only one of them is functional (23). An alginate lyase containing two functional catalytic domains has not been reported so far.

The synergy of alginate lyases has attracted increasing attention since it plays important roles in microbial alginate utilization and biotechnological applications. Alginate lyases often coexist in the same genome and are thus thought to work synergistically to cause the fast and complete digestion and utilization of alginate by bacteria (24). In addition, the synergistic effect of alginate lyases can be used to efficiently degrade alginate in brown algae to produce ethanol (25). The so-far reported synergistic mechanisms of alginate lyases are all limited to those between multiple lyases. The synergy between different catalytic domains of one single lyase has not been studied (6, 26).

In this study, an alginate lyase AlyC6’ with two catalytic domains was identified from a marine alginate-degrading bacterium, Vibrio sp. NC2, that was isolated from a Colpomenia sample. Its two catalytic domains, CD1 and CD2, were both annotated as alginate lyase domains of the PL7 family, but without sequence similarity. The alginolytic activity of the two domains was verified to be functional in AlyC6’, and their properties were characterized in detail. The results indicated that the different properties of the two domains enable them to synergize intramolecularly in alginate degradation. Moreover, bioinformatics analysis indicated that diverse alginate lyases with multiple alginate lyase domains are widespread and may be a strategy for bacteria to promote the degradation of alginate. The results are helpful for understanding bacterial alginate degradation and shed light on designing new multimodular alginate lyases for efficient alginate degradation.

RESULTS AND DISCUSSIONS

Sequence analysis of AlyC6’.

The gene alyC6’ in the genome of Vibrio sp. NC2, an alginate-degrading strain isolated from a Colpomenia sample, was predicted to encode an alginate lyase. alyC6’ is 1,743 bp in length and encodes a putative alginate lyase (AlyC6’) of 580 amino acid residues with a predicted 16-residue signal peptide (Fig. 1A). According to the blast result against the NCBI conserved domain database, AlyC6’ contains two alginate lyase domains, the CD1 domain (residues 56 to 285) and the CD2 domain (residues 294 to 579) (Fig. 1A). Phylogenetic analysis showed that CD1 and CD2 belong to subfamily 6 and subfamily 3 of the PL7 family, respectively (Fig. 1B). However, the two domains share quite low sequence similarity (13.9%), suggesting that they may have distinct characteristics. AlyC6’ exhibits the highest sequence identity (95.17%) with AlyA, an alginate lyase with two annotated alginate lyase domains, one of which is functional (23). The CD1 and CD2 domains of AlyC6’ share 98.26% and 93.71% sequence similarity with the N- and C-terminal domains of AlyA, respectively.

FIG 1.

Sequence analysis of the alginate lyase AlyC6’ from Vibrio sp. NC2. (A) Schematic domain diagram of the alginate lyase AlyC6’ with other PL7 alginate lyases. AlyA (GenBank: EAP94921.1) and AlyD (GenBank: EAP94925.1) are from Vibrio splendidus 12B01, Algb (GenBank: KM507331) from Vibrio sp. W13, and AlyB (PDB code 5ZU5) from Vibrio spendidus OU02. The signal peptide was predicted by the SignalP 5.0 server. The domain composition was analyzed by blasting at the Conserved Domain database. CBM32, the carbohydrate-binding module family 32 domain. (B) Phylogenetic analysis of the CD1 domain, the CD2 domain, and the characterized PL7 alginate lyases. The phylogenetic tree was constructed using the MEGA 7 software via the neighbor-joining method. Bootstrap analysis of 1,000 replicates was conducted.

Determination of the alginolytic activity of AlyC6’ and its two alginate lyase domains.

To determine whether AlyC6’ and its two annotated catalytic domains have alginolytic activity, we constructed two truncated mutants of AlyC6’, ΔCD2 (residues 56 to 290) lacking the CD2 domain and ΔCD1 (residues 290 to 580) lacking the CD1 domain (Fig. 2A and B). Then, the alginolytic activity of AlyC6’, ΔCD1, and ΔCD2 were determined. As shown in Fig. 2C, all the proteins showed noticeable activity toward sodium alginate, suggesting that both CD1 and CD2 are likely functional in AlyC6’.

FIG 2.

Determination of the alginolytic activity of AlyC6’, AlyC6’-CD1, and AlyC6’-CD2. (A) Domain structure of the recombinant AlyC6’ and its mutants. (B) SDS-PAGE analysis of the purified AlyC6’ and its mutants. (C) Determination of the alginolytic activity of AlyC6’ and its mutants by HPLC. The reactions were all carried out at 30°C for 10 min in the mixture containing enzyme, 2 mg/mL sodium alginate, 1.0 M NaCl, and 50 mM Tris-HCl (pH 8.0). The activity was determined by HPLC and monitored at 210 nm using a UV detector. The control was treated with preheated inactivated lyases. Saturated mannuronate oligosaccharides from DP1 to DP6 were taken as the standards. DP, degree of polymerization. (D) Multiple sequence alignment of the CD1 and CD2 domains with other PL7 alginate lyases whose catalytic mechanisms have been revealed. Black stars indicate the catalytic amino acid residues.

To further verify that both domains play a role in the full-length enzyme, we determined the alginolytic activity of one single domain in AlyC6’ by abolishing the activity of the other domain. As reported, a conserved histidine and a conserved tyrosine usually act as the two catalytic residues in the PL7 alginate lyases (27). Based on the multiple sequence alignment of the CD1 and CD2 domains with other characterized PL7 alginate lyases, H155 and Y266 are likely the catalytic residues of the CD1 domain, and H438 and Y554 are likely those of the CD2 domain (amino acid residues were labeled according to their positions within the AlyC6’ sequence) (Fig. 2D). We then constructed the mutants H155A_Y266A of ΔCD2 (named ΔCD2-H155A_Y266A) and H438A_Y554A of ΔCD1 (named ΔCD1-H438A_Y554A) and found that they both completely lost alginolytic activity (Fig. 2A, to C). Afterward, the mutants, H155A_Y266A of AlyC6’ (named AlyC6’-CD2), which lost the CD1 domain activity, and H438A_Y554A of AlyC6’ (named AlyC6’-CD1), which lost the CD2 domain activity, were constructed (Fig. 2A and B). As shown in Fig. 2C, both AlyC6’-CD1 and AlyC6’-CD2 have alginolytic activity, demonstrating that the CD1 and CD2 domains can degrade alginate independently in AlyC6’. Altogether, the results showed that AlyC6’ is a lyase with two functional alginate lyase domains.

To date, two alginate lyases containing two catalytic domains have been reported. One is Algb; however, the activity of its two catalytic domains has not been determined (22). The other is AlyA, and only its C-terminal catalytic domain was shown to have alginolytic activity (23). In contrast, the above results showed that the two catalytic domains of AlyC6’ both have alginolytic activity. Therefore, AlyC6’ is a novel alginate lyase with two alginate lyase domains, both of which are active in the full-length enzyme.

Biochemical characterization of AlyC6’, AlyC6’-CD1, and AlyC6’-CD2.

(i) The effects of enzymatic reaction conditions on the activity of AlyC6’, AlyC6’-CD1, and AlyC6’-CD2. AlyC6’, AlyC6’-CD1, and AlyC6’-CD2 all had the maximum activity at 30°C, retaining more than 56% of their maximum activities at 20 to 30°C. When the temperature reached 50°C or higher, more than 85% of their maximum activities were lost (Fig. 3A). These enzymes all showed the highest activity at pH 8.0 and retained at least 83% activity at pH 9.0 (Fig. 3B). In addition, their activities were all activated by NaCl, reaching the highest activity at 1.0 M NaCl and retaining more than 83% of the maximum enzyme activity at the NaCl concentrations ranging from 0.5 M to 1.5 M (Fig. 3C).

FIG 3.

The effects of enzymatic reaction conditions on the activity of AlyC6’, AlyC6’-CD1, and AlyC6’-CD2 toward sodium alginate. (A) The effect of temperature on enzyme activities. Experiments were conducted at 10 to 50°C for 10 min in a 200-μL reaction mixture containing enzyme, 2 mg/mL sodium alginate, 50 mM Tris-HCl (pH 8.0), and 1.0 M NaCl. (B) The effect of pH on enzyme activities. A 200-μL reaction mixture containing enzyme, 2 mg/mL sodium alginate in 1.0 M NaCl, and 50 mM Britton-Robinson buffer ranging from pH 6.0 to 10.0 was incubated at 30°C for 10 min. (C) The effect of NaCl concentration on enzyme activities. A 200-μL reaction mixture containing enzyme, 2 mg/mL sodium alginate in 50 mM Tris-HCl (pH 8.0), and NaCl at the concentrations from 0 to 2.375 M was incubated at 30°C for 10 min. The concentrations of AlyC6’, AlyC6’-CD1, and AlyC6’-CD2 in the reaction mixture were 0.72 μg/mL, 1.15 μg/mL, and 4.60 μg/mL, respectively. The graphs show data from triplicate experiments (mean ± standard deviation [SD]).

The enzymatic reaction conditions of alginate lyases are usually related to the living environment of their source strains. For example, the PL7 alginate lyase AlyA1 from the marine bacterium Zobellia galactanivorans showed its highest activity at 30°C, pH 7.0, and 200 mM NaCl and retained more than 60% of the maximum activity at the range of 20 to 40°C and 0.1 to 0.8 M NaCl (28). The PL6 alginate lyase AlyPB1 and PL15 alginate lyase AlyPB2 from the marine bacterium Photobacterium sp. FC615 had the highest activity at 20 to 30°C and pH 8.0. They lost more than 80% of the maximum activity when the temperature reached 40°C or higher (24). The PL7 alginate lyase AlyC3 from Psychromonas sp. C-3 isolated from the Arctic brown alga Laminaria exhibited the highest activity at pH 8.0 and 20°C, and retained 48.2% of its maximum activity at 1°C (27). In summary, marine alginate lyases have higher activities at the range of 20 to 40°C, pH 7.0 to 9.0, and 0 to 1 M NaCl, reflecting their adaption to the marine environment. Similarly, though the CD1 domain always showed a much higher activity toward sodium alginate than the CD2 domain under the same reaction conditions, temperature, pH, and NaCl concentrations had similar effects on the activities of AlyC6’, AlyC6’-CD1, and AlyC6’-CD2. All the enzymes had the highest activity at 30°C, pH 8.0, and 1.0 M NaCl, which ensures their high activity under the low-temperature, alkalescent and saline marine environment.

(ii) The substrate specificities of AlyC6’, AlyC6’-CD1, and AlyC6’-CD2. To determine their substrate specificity, we measured the activities of AlyC6’, AlyC6’-CD1, and AlyC6’-CD2 toward different alginate substrates, including PM, PG, PMG, and sodium alginate. AlyC6’-CD1 showed high activities toward PM, PMG, and sodium alginate, but almost no activity toward PG; contrarily, AlyC6’-CD2 showed the highest activity toward PG, but almost no activity toward PM (Fig. 4), which indicated that AlyC6’-CD1 and AlyC6’-CD2 have complementary substrate selectivity. AlyC6’ showed a similar substrate preference with AlyC6’-CD1, probably due to the major role of AlyC6’-CD1 in AlyC6’. Remarkably, AlyC6’ exhibited detectable, although not high, activity toward PG (Fig. 4), which should benefit from the substrate preference for PG of AlyC6’-CD2. These results demonstrated that the coexistence of the CD1 and CD2 domains makes AlyC6’ significantly active toward various alginate substrates.

FIG 4.

Substrate specificity of AlyC6’, AlyC6’-CD1, and AlyC6’-CD2 toward PM, PG, PMG, and sodium alginate. The reactions were all carried out at 30°C for 10 min in the mixture containing enzyme (AlyC6’, 0.72 μg/mL; AlyC6’-CD1, 1.15 μg/mL; AlyC6’-CD2, 4.60 μg/mL), 2 mg/mL substrate, 50 mM Tris-HCl (pH 8.0), and 1.0 M NaCl. The increase in the absorbance at 235 nm in the mixture was monitored. The graph shows data from triplicate experiments (mean ± standard deviation [SD]).

(iii) The minimal substrates of AlyC6’, AlyC6’-CD1, and AlyC6’-CD2. The minimal substrates of AlyC6’, AlyC6’-CD1, and AlyC6’-CD2 were studied with the preferred alginate homopolymer substrate of each enzyme as the substrate. As shown in Fig. 5, AlyC6’-CD2 could not degrade trimer into dimer but could degrade tetramer into smaller products (the generated unsaturated monomer was hard to be observed due to its instability), indicating that the minimal substrate of AlyC6’-CD2 is a tetramer. AlyC6’-CD1 could degrade trimer into dimer but could not degrade dimer into monomer, indicating that the minimal substrate of AlyC6’-CD1 is a trimer. Therefore, the CD1 domain has a smaller minimal substrate than the CD2 domain. Same as AlyC6’-CD1, the minimal substrate of AlyC6’ is a trimer, likely due to the degradation of the trimer by AlyC6’-CD1.

FIG 5.

Analysis of the minimal substrates of AlyC6’, AlyC6’-CD1, and AlyC6’-CD2. All the reactions were carried out toward 2 mg/mL substrate at 30°C for 24 h in the buffer containing 50 mM Tris-HCl (pH 8.0) and 1.0 M NaCl. AlyC6’ and AlyC6’-CD1 reacted with mannuronate oligomers, while AlyC6’-CD2 reacted with guluronate oligomers based on their substrate specificities. The degrees of polymerization (DPs) of the oligomer substrates were from 2 to 4. The degradation products were analyzed by HPLC on a Superdex Peptide 10/300 GL column using 0.2 M ammonium hydrogen carbonate as the running buffer. Elution was monitored at 210 nm using a UV detector. 2M, 3M, and 4M represent di-, tri-, and tetra-mannuronate, respectively. 2G, 3G, and 4G represent di-, tri-, and tetra-guluronate, respectively.

(iv) Kinetic parameters of AlyC6’, AlyC6’-CD1, and AlyC6’-CD2. The kinetic parameters of AlyC6’, AlyC6’-CD1, and AlyC6’-CD2 toward sodium alginate were analyzed (Table 1). The K_m value of AlyC6’-CD1 was noteworthily higher than that of AlyC6’-CD2, indicating that the affinity for sodium alginate of AlyC6’-CD2 was higher than that of AlyC6’-CD1; the k_cat/K_m value of AlyC6’-CD1 was higher than that of AlyC6’-CD2, indicating that AlyC6’-CD1 had a higher catalytic efficiency. Strikingly, though the K_m value of AlyC6’ was close to that of AlyC6’-CD1, the k_cat/K_m value of AlyC6’ was remarkably higher than the sum of those of AlyC6’-CD1 and AlyC6’-CD2, suggesting that the two domains in AlyC6’ may have synergy in alginate degradation.

TABLE 1.

Kinetic parameters of AlyC6’-CD1 and AlyC6’-CD2 toward sodium alginate

Enzyme	Km (mg/mL)	Vmax (U/mg)	Kcat (s−1)	Kcat/Km (s−1 mg−1 mL)
AlyC6’	2.12 ± 0.17	7793.94 ± 235.61	249.64 ± 7.55	117.75
AlyC6’-CD1	2.09 ± 0.17	4664.05 ± 253.98	132.17 ± 7.20	63.24
AlyC6’-CD2	0.51 ± 0.02	594.39 ± 12.42	10.22 ± 0.21	20.04

Synergy of the CD1 and CD2 domains of AlyC6’ in alginate degradation.

In theory, the CD2 domain cannot degrade the M-M bonds in alginate, and thus its degradation products are rich in the uncleaved M-M bonds that can be cleaved by the CD1 domain. Moreover, the CD1 domain can degrade smaller substrates compared with the CD2 domain, and thus can further degrade oligomeric products generated by the CD2 domain. Based on this analysis, it is tempting to hypothesize that AlyC6’-CD1 can actually degrade the oligomers produced by AlyC6’-CD2 significantly. Thus, we collected the products of AlyC6’-CD1 reacting with sodium alginate for 1 h (P_CD1) and the products of AlyC6-CD2 reacting with sodium alginate for 1 h (P_CD2), respectively. Apparently, P_CD1 and P_CD2 were not the final products of the reactions. Afterwards, AlyC6’-CD1 was mixed with P_CD1 or P_CD2, and the products from different reaction times were then analyzed. As expected, AlyC6’-CD1 degraded P_CD1 quite slowly, and only a small quantity of oligomers at degrees of polymerization (DP) of 4 and 5 were further degraded (Fig. 6A). In contrast, AlyC6’-CD1 significantly degraded P_CD2 into smaller products. As time increased, tetrasaccharides and larger saccharides gradually decreased, and disaccharides and trisaccharides gradually increased (Fig. 6A). These results implied that when AlyC6’-CD1 and AlyC6’-CD2 degrade alginate, AlyC6’-CD1 can effectively degrade oligosaccharides produced by AlyC6’-CD2, which is conducive to further degradation of the generated oligomeric products into products at smaller DPs.

FIG 6.

The synergy of the CD1 and CD2 domains in alginate degradation. (A) Time course treatment of AlyC6’-CD1 reacting with the products generated by AlyC6’-CD1 and AlyC6’-CD2. AlyC6’-CD1 and AlyC6’-CD2 first reacted with sodium alginate in the reaction mixture containing 2 mg/mL sodium alginate, 1.0 M NaCl, and 50 mM Tris-HCl (pH 8.0) at 30°C for 1 h, respectively. Then, the enzyme, the substrate, and products with high molecular weights (>3 kDa) were removed and the filtrate containing oligosaccharide products was concentrated to 110 μg/mL. Afterwards, AlyC6’-CD1 was mixed with the products generated by AlyC6’-CD1 or AlyC6’-CD2 for different times, and the resultant products were detected by HPLC. The degradation products of AlyC6’-CD1 and AlyC6’-CD2 toward sodium alginate were taken as the control groups. (B) Final degradation products of AlyC6’, AlyC6’-CD1, AlyC6’-CD2, and an enzyme mixture containing AlyC6’-CD1 and AlyC6’-CD2 at a molar ratio of 1:1 toward sodium alginate (SA). A 200-μL reaction mixture containing enzyme(s), 2 mg/mL sodium alginate in 50 mM Tris-HCl (pH 8.0), and 1.0 M NaCl was incubated at 30°C for 24 h. DP1, DP2, DP3, DP4, DP5, and DP6, representing saturated monosaccharide, disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, and hexasaccharide, respectively, were taken as the standards. UDP2, UDP3, UDP4, UDP5, UDP6, and UDP7 represent unsaturated disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide, and heptasaccharide, respectively. (C) The synergistic effect of the CD1 and CD2 domains on alginate decomposition. Each enzyme (2.2 μg/mL) or the enzyme mixture containing 2.2 μg/mL AlyC6’-CD1 and 2.2 μg/mL AlyC6’-CD2 was incubated with 2 mg/mL sodium alginate in the buffer containing 50 mM Tris-HCl (pH 8.0) and 1.0 M NaCl at 30°C for 10 min.

We further investigated the final degradation products of AlyC6’, AlyC6’-CD1, AlyC6’-CD2, and an enzyme mixture containing AlyC6’-CD1 and AlyC6’-CD2 at a molar ratio of 1:1. The majority of the final degradation products of AlyC6’-CD1 toward sodium alginate were dimers and trimers, and oligomers larger than trimers, e.g., tetramers, pentamers, hexamers, and heptamers, were also generated (Fig. 6B). The major products of AlyC6’-CD2 were oligomers at DP3 to DP5, and dimers and oligomers larger than pentamers (including hexamers and heptamers) were also produced (Fig. 6B). Among the products of the enzyme mixture containing AlyC6’-CD1 and AlyC6’-CD2, the oligosaccharides at DP2 to DP5 were predominant, with a small number of hexamers (Fig. 6B). Heptamers, which were observed in both the degradation products of AlyC6’-CD1 and those of AlyC6’-CD2, were almost absent from the products of the enzyme mixture (Fig. 6B), indicating that the combination of AlyC6’-CD1 and AlyC6’-CD2 can degrade alginate into small oligosaccharide products. The final products of AlyC6’ contained many dimers and trimers and a small number of tetramers and pentamers. Of note, no saccharides larger than pentamers were observed among the products of AlyC6’ (Fig. 6B). Moreover, AlyC6’ produced a higher proportion (84.3%) of small oligosaccharide products (dimers and trimers) and a lower proportion (15.7%) of tetramers and pentamers compared to the mixture of AlyC6’-CD1 and AlyC6’-CD2 (71.5% for dimers and trimers, 26.9% for tetramers and pentamers), based on the peak area of the products in the gel filtration chromatogram (Fig. 6B). These results indicated that the coexistence of the CD1 and CD2 domains in AlyC6’ synergistically degraded alginate into small oligosaccharide products, and that the synergy of the two catalytic domains in one polypeptide was more efficient than that of the mixture containing two separate catalytic domains.

In addition, the synergy of the CD1 and CD2 domains in alginate degradation was also analyzed by enzymatic activity assays. As shown in Fig. 6C, the activity of the mixture containing AlyC6’-CD1 and AlyC6’-CD2 at a molar ratio of 1:1 was lower than that of AlyC6’ but close to that of AlyC6’-CD1 plus that of AlyC6’-CD2. The result further demonstrated that the CD1 and CD2 domains can synergize intramolecularly rather than intermolecularly (29), and also implied the necessity of the two catalytic domains within the same polypeptide for decomposing alginate, which was consistent with the degradation products analysis results. The proximity of two catalytic domains with synergistic effects enables the products produced by one domain to be rapidly bound to and further degraded by the other domain. If the two domains are separate rather than tethered in a polypeptide, as is the case between multiple enzymes with a single catalytic domain, their synergy may be diminished.

Overall, the CD1 and CD2 domains showed conspicuous synergy in alginate degradation. The two domains have complementary substrate scopes. The highest activity of the CD2 domain toward PG compensates for the absent activity of the CD1 domain toward PG, enabling AlyC6’ to degrade all types of glycosidic bonds in alginate. Moreover, the minimal substrate of the CD1 domain is smaller than that of the CD2 domain, which facilitates the further degradation of the products of the CD2 domain by the CD1 domain. All the different properties above between the two domains are helpful for alginate degradation. Furthermore, the two domains may also act synergistically due to their different kinetic properties. The released oligomers degraded by the CD2 domain probably increase the substrate concentration around the catalytic center of the adjacent CD1 domain, which is favorable for alginate degradation by the CD1 domain at a higher catalytic rate.

Synergistic effects of alginate lyases have been reported by many studies. Lu et al. reported that an endolytic alginate lyase and an exolytic alginate lyase from Photobacterium sp. FC615 had a strong synergistic effect on the saccharification of alginate (24). Three exolytic oligoalginate lyases from Vibrio splendidus have been shown to have complementary substrate scopes and temperature and pH adaptations (26). PsAlg7A and PsMan8A from the marine fungus Paradendryphiella salina exhibited a clear synergistic action for the complete depolymerization of PM at the specific pH value (6). Alekseeva et al. reported that the mixture of three intracellular alginate lyases of Pseudoalteromonas citrea KMM 3297 showed synergy while acting on the polymeric alginate (30). However, they all focused on the synergy of multiple alginate lyases equipped with a single catalytic domain.

Compared with that of multiple alginate lyases, the synergistic effect of the two domains of a single enzyme showed its enticing advantages, namely, the adjacency of the two domains enables them to exert a more pronounced synergy. Obviously, this kind of multimodular alginate lyase will be beneficial for bacteria to degrade alginate. Meanwhile, the synergy between different catalytic domains of one alginate lyase can be used to guide the design of new enzymes for efficient alginate depolymerization, since the alginate degradation in industry has always been accomplished by multienzyme cocktails.

Distribution of alginate lyases with multiple alginate lyase domains.

By analyzing the alginate lyase sequences in the CAZy database, we found that there are various alginate lyases with multiple catalytic domains (MCD-ALs) and that their domain compositions are different (Fig. 7). Among them, alginate lyases with two PL6 domains are the most abundant. Actually, Xu et al. reported that the PL6 alginate lyase AlyGC consists of two PL6 domains that exhibit a similar β-parallel structure (31), although only the N-terminal PL6 domain has alginolytic activity. Considering that the N-terminal PL7 domain of AlyA has no activity but its C-terminal PL7 domain does, whereas both the PL7 domains of AlyC6’ have alginolytic activity, we speculated that some alginate lyases may have two functional PL6 domains, which requires further investigation. There are also many alginate lyases with two PL7 domains, most of which are from Pseudomonadota, especially Vibrionales, demonstrating that AlyC6’ from Vibrio sp. NC2 is a typical alginate lyase with two PL7 domains. In addition, several alginate lyases with both PL6 and PL7 domains were found. These proteins contain at least three alginate lyase domains, that is, an enzyme has more than one PL6 or PL7 domain. Moreover, two alginate lyases with two PL5 domains, one lyase consisting of a PL7 domain and a PL18 domain and one lyase comprising two PL31 domains, were found from bacteria. Additionally, one enzyme containing two PL14 alginate lyase domains was found from Oikopleura dioica, a ubiquitous planktonic chordate, indicating that eukaryote may also produce MCD-ALs. Taking the protein sequences of the MCD-ALs searched from the CAZy database as the templates, we further investigated their distribution in the metagenomes (MetaG) and metatranscriptomes (MetaT) from the nonpolar Tara Ocean data set (Fig. 7). The results of macro-omics analyses were relatively congruent with those of the CAZy database. Enzymes comprising two PL6 domains are the most abundant, followed by those containing two PL7 domains and those containing PL6 and PL7 domains. Fewer MCD-ALs are with other domain compositions.

FIG 7.

Distribution of alginate lyases with multiple alginate lyase domains. The domain compositions of alginate lyases with multiple catalytic domains were analyzed by the dbCAN server and are shown as a schematic boxed-in dashed line at the bottom. The taxonomic compositions of alginate lyases are displayed at the order and phylum levels. CAZy, carbohydrate-active enZYmes database; MetaG, metagenomes; MetaT, metatranscriptomes; PL, polysaccharide lyase.

Different alginate lyase domains of MCD-ALs may have different biochemical properties, such as different minimal substrate and kinetic parameters, and complementary enzymatic properties, including enzymatic reaction scopes, substrate preferences, and modes of action. By combining multiple alginate lyase domains in a polypeptide, enzymes are capable of remarkably degrading alginate by a more effective synergistic effect of these domains, just as in the case of AlyC6’.

Various bacteria can produce MCD-ALs, including those of Pseudomonadota, Actinobacteria, Firmicutes, Bacteroidetes, Cyanobacteria, Verrucomicrobia, Spirochaetes, Fibrobacteres, and Planctomycetes (Fig. 7), indicating that MCD-ALs may be widespread. Analyses of the CAZy, MetaG, and MetaT data sets all showed that among the MCD-ALs, most are from Pseudomonadota and Bacteroidetes. Furthermore, Alteromonadales of Pseudomonadota and Flavobacteriales of Bacteroidetes have the most types of domain compositions and largest numbers of MCD-ALs. Alteromonadales and Flavobacteriales are known to be versatile degraders of marine polysaccharides, including alginate (32). Wietz et al. showed that the addition of 0.001% alginate to seawater from the Patagonian continental shelf induced a strong increase in Alteromonadaceae that could reach 80% final relative abundance (33). Thomas et al. traced ¹³C derived from alginate into specific bacterial incorporators and quantified the uptake activity at the single-cell level, and their results revealed that only a few Flavobacteriaceae and Alteromonadales taxa incorporated ¹³C from alginate into their biomass, accounting for most of the carbon assimilation (32). Therefore, it appears that bacteria, such as Flavobacteriaceae and Alteromonadales, may promote their degradation of alginate by secreting MCD-ALs that are particularly powerful in alginate degradation.

Conclusion.

Herein, a novel alginate lyase, AlyC6’, with two functional alginate lyase domains from the marine bacterium Vibrio sp. NC2, was biochemically characterized. The two domains show a significant synergistic degradation effect on alginate based on their complementary substrate preference and different minimal substrate and kinetic parameters. The intramolecular synergy of the two domains in the full-length enzyme facilitates the efficient and complete alginate degradation and the release of small oligomers. In addition, MCD-ALs with diverse domain compositions are widespread in nature, and their secretion may be a strategy for bacteria to promote the degradation of alginate. The results provide new insights on alginate lyases and bacterial alginate degradation and are helpful to guide design of new enzymes for efficient alginate depolymerization.

MATERIALS AND METHODS

Materials and strains.

Sodium alginate derived from brown algae was purchased from Sigma (USA). PM and PG (6 to 8 kDa) were purchased from Zzstandard (China). Oligosaccharide substrates were purchased from BZ Oligo Biotech Co., Ltd. (China). PMG was prepared as previously described (5). Other chemicals and reagents used in this study are of analytical grade. Vibrio sp. NC2, previously isolated from a Colpomenia sample collected from coastal seawaters in Shandong Province, China, was preserved in our lab. Escherichia coli strains were purchased from Tsingke (China) and cultured in Lysogeny broth (LB) medium at 37°C.

Bioinformatics analysis.

The genomic DNA of Vibrio sp. NC2 was shotgun-sequenced on the Illumina Hiseq sequencing platform (Majorbio, China). The putative alginate lyases in Vibrio sp. NC2 were predicted by the dbCAN meta server (https://bcb.unl.edu/dbCAN2/blast.php) (34) based on the genome of Vibrio sp. NC2. The residues encoding a putative signal peptide were predicted by the SignalP 5.0 server (https://services.healthtech.dtu.dk/service.php?SignalP-5.0) (35). The conserved domains analysis was performed by blasting at the Conserved Domain database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) (36). The putative catalytic residues were analyzed by multiple sequence alignment carried out by ClustalW (37). The multiple sequence alignment figure was prepared using the ESPript program (38).

MCD-ALs were manually retrieved based on domain analysis of the alginate lyases of the PL5, -6, -7, -8, -14, -15, -17, -18, -31, -32, -34, -36, -39, and -41 families by using the dbCAN meta server (34). The taxonomic classification of each sequence was searched against the nonredundant protein sequences (nr) database using the GenBank number. Hidden Markov models (HMM) were created using protein sequences of the enzymes with the same domain composition searched from the CAZy database. The MCD-ALs with different domain compositions in the MetaG and MetaT from the nonpolar Tara Ocean data set were obtained using the created hmm files. The distribution of MCD-ALs was visualized by Sankey diagrams using the HIPLOT online website (https://hiplot.org) (39, 40).

Gene cloning and mutagenesis.

The alyC6’ gene (GenBank: WP_016791256.1) was amplified from the genomic DNA of Vibrio sp. NC2 via PCR and cloned into the vector pET-22b between the restriction sites NdeI and XhoI along with a C-terminal His tag. Site-directed and truncated mutations on alyC6’ were conducted by using a QuikChange kit (Agilent, USA).

Protein expression and purification.

Recombinant AlyC6’ and its mutants were expressed in E. coli BL21(DE3). The cells were cultured at 18°C for 16 h in LB broth containing 100 μg/mL ampicillin under the induction of 0.3 mM isopropyl-D-1-thiogalactopyranoside (IPTG). Cells were collected and disrupted by a JN-02C French press (JNBIO, China) in the buffer containing 50 mM Tris-HCl (pH 8.0) and 100 mM NaCl. After centrifugation at 15,000 × g for 1 h at 4°C, the recombinant proteins were first purified by Ni-affinity chromatography (Qiagen, Germany) and then purified by gel filtration chromatography on a Superdex 200 column (GE Healthcare, USA) in the buffer containing 10 mM Tris-HCl (pH 8.0) and 100 mM NaCl. The target protein was collected, and the protein concentration was determined by the bicinchoninic acid (BCA) protein assay kit (Thermo, USA) with bovine serum albumin (BSA) as the standard.

Determination of alginolytic activity.

To determine the alginolytic activity of AlyC6’ and its mutants, the enzymes were individually reacted in a 200-μL reaction system, which contained 2 mg/mL sodium alginate, 50 mM Tris-HCl (pH 8.0), 1.0 M NaCl, and enzyme (AlyC6’, 0.72 μg/mL; AlyC6’-CD1, 1.15 μg/mL; AlyC6’-CD2, 4.60 μg/mL; ΔCD2, 0.61 μg/mL; ΔCD1, 2.32 μg/mL; ΔCD2-H155_Y266, 15.66 μg/mL; ΔCD1-H438_Y554, 57.80 μg/mL). After the reaction mixture was incubated at 30°C for 10 min, the reaction was terminated by an addition of 0.4 M trichloroacetic acid (TCA) into the mixture. The alginate lyase activity of the enzymes was determined by high performance liquid chromatography (HPLC) on a Superdex Peptide 10/300 GL column (GE Healthcare, USA) at a flow rate of 0.3 mL/min using 0.2 M ammonium hydrogen carbonate as the running buffer. Elution was monitored at 210 nm using a UV detector. LabSolutions software was used for online monitoring and data analysis.

Biochemical characterization of alginate lyases.

The activity of the alginate lyases AlyC6’, AlyC6’-CD1, and AlyC6’-CD2 were measured by the UV absorption spectrometry method (41). The reaction system (200 μL) contained 2 mg/mL substrate, 50 mM Tris-HCl (pH 8.0), 1.0 M NaCl, and enzyme (AlyC6’, 0.72 μg/mL; AlyC6’-CD1, 1.15 μg/mL; AlyC6’-CD2, 4.60 μg/mL). The mixture was incubated at 30°C for 10 min. The reaction was then terminated by boiling the mixture for 10 min. The increase in the absorbance at 235 nm (A₂₃₅), resulting from the release of an unsaturated uronic in the mixture, was monitored. One unit (U) of enzyme activity was defined as the amount of enzyme required to cause an increase of 0.1 at 235 nm per minute. The optimum temperature for AlyC6’, AlyC6’-CD1, and AlyC6’-CD2 activity was determined at a range of 20 to 50°C at pH 8.0. The optimum pH for AlyC6’, AlyC6’-CD1, and AlyC6’-CD2 activity was determined at 30°C in the Britton-Robinson (B-R) buffer ranging from pH 6.0 to 10.0. B-R buffer was prepared with boric acid, acetic acid, and phosphoric acid, all with the final concentration of 0.04 M in an aqueous solution, and was adjusted to different pH with 0.2 M NaOH. The effect of NaCl concentrations on AlyC6’, AlyC6’-CD1, and AlyC6’-CD2 activity was determined at NaCl concentrations ranging from 0 to 2.375 M. Substrate specificity of AlyC6’, AlyC6’-CD1, and AlyC6’-CD2 was tested by measuring their activity toward PM, PG, PMG, and sodium alginate.

The kinetic parameters of AlyC6’, AlyC6’-CD1. and AlyC6’-CD2 for depolymerization of sodium alginate were determined as described previously (42). For each enzyme, initial velocities were quantified on sodium alginate (0.25 to 6.75 mg/mL) under their optimal reaction conditions. The A₂₃₅ was recorded to quantify the concentrations of the oligoalginate using a molar extinction coefficient of ϵ = 6150 M⁻¹cm⁻¹ (43). The kinetic parameters K_m and V_max were determined from nonlinear regression fitting of the Michaelis-Menten equation using the Origin 8.5 software. The catalytic constant (k_cat) was calculated by the ratio of V_max to enzyme concentration (E) (42).

Degradation products analysis.

The degradation reaction of AlyC6’, AlyC6’-CD1, AlyC6’-CD2, and an enzyme mixture containing AlyC6’-CD1 and AlyC6’-CD2 at a molar ratio of 1:1 toward sodium alginate or oligosaccharide standards (2 mg/mL) was carried out in the buffer containing 50 mM Tris-HCl (pH 8.0) and 1.0 M NaCl at 30°C for 24 h. The final concentration of the enzyme(s) in the reaction mixture was 0.3 nM. The reaction was terminated by an addition of 0.4 M TCA into the mixture.

To analyze the degradation products of AlyC6’-CD1 on P_CD1 or P_CD2, 20 μL AlyC6’-CD1 (4.5 mg/mL) or AlyC6’-CD2 (10.9 mg/mL) was added into a 200-μL reaction mixture containing 2 mg/mL sodium alginate, 1.0 M NaCl, and 50 mM Tris-HCl (pH 8.0), and the mixtures were incubated at 30°C for 1 h. Then, the enzyme, the substrate, and products with high molecular weights (MWs) were removed by ultrafiltration with a 3-kDa ultrafiltration tube. The filtrate containing oligosaccharides was concentrated by rotary evaporation. The concentration of oligosaccharides was determined by the DNS method and then adjusted to 110 μg/mL. Afterwards, these two kinds of oligosaccharide products, namely, P_CD1 or P_CD2, were further reacted with AlyC6’-CD1 at 30°C for different times, respectively.

All the degradation products were analyzed by HPLC according to the method described in the section Determination of Alginolytic Activity.