Sequence Analysis
The open reading frame (ORF) of AlyPL17 consists of 2085bp encoded a putative alginate lyase composed of 695 amino acids with a theoretical molecular mass of 78.07 kDa. As shown in Fig. 2A, the full-length enzyme contains two domains, an alginate lyase domain (designed as AlyPL17-N, Lys37-Gln265) and a heparinase II/III-like domain (designed as AlyPL17-C, Arg357-Phe528). The phylogenetic tree was constructed and it exhibited AlyPL17 clusters with several alginate lyases of PL17 family. Based on the alignment of protein sequence, AlyPL17 has the highest sequence identity (45%) with OAL from Stenotrophomonas maltophilia KJ-2 (GenBank accession no. AGM38186.2) (Shin et al., 2015), followed by Alg17c (44%) from Saccharophagus degradans 2–40 (GenBank accession no. ABD82539.1) (Park et al., 2014). Furthermore, several key amino acid residues of PL17 family are highly conserved in AlyPL17 such as N177, H178, Y234,Y426 in active sites; N125, R236, Y237, H389, R414 in the substrate binding sites.
Based on the homologous structure of Alg17c (PDB: 4NEI) from Saccharophagus degradans 2–40, three-dimensional models of AlyPL17 and its truncated mutants were constructed by PHYRE2. The sequence identity between AlyPL17 and Alg17c was high (44%). Accordingly, the protein model was successfully constructed with 100% confidence. As shown in Fig. 2B, AlyPL17 harbors two domains, AlyPL17-N is folded as a (α/α)6 toroid fold, and AlyPL17-C is folded as a β-sandwich jellyroll with two anti-parallel β sheets (Fig. 2C, 2D).
Expression and Purification of AlyPL17 and Its Truncated Mutants
After heterologously expressed and purified, the recombinant AlyPL17, AlyPL17-N and AlyPL17-C were obtained and then analyzed by SDS-PAGE. In Fig. 3, three clear bands represented the molecular mass of purifiedAlyPL17, AlyPL17-N and AlyPL17-C can be observed, which are consistent with the predicted molecular mass of 78.07 kDa, 39.22 kDa and 39.78 kDa, respectively. The molecular mass (Mr) of other PL17 alginate lyases varied from 75 kDa to 86 kDa. For example, OalB from Vibrio splendidus 12B01 (Jagtap et al., 2014), Alg17c from Saccharophagus degradans 2–40 (Park et al., 2014) and Oal17A from Vibrio sp. W13 (Lyu et al., 2018) possess the molecular mass of 83 kDa, 79 kDa and 81 kDa, respectively.
Three different substrates (0.5% sodium alginate, 0.5% polyM and 0.5% polyG) were applied to determine the activities of AlyPL17 and its truncated mutants. As shown in Table 1, the full-length enzyme AlyPL17 exhibited higher activities towards polyM (861.62 ± 1.53 U/mg) than towards sodium alginate (808.53 ± 2.23 U/mg), and polyG (802.62 ± 2.45 U/mg). To the best of our knowledge, AlyPL17 has relatively higher enzymatic activities than most PL17 family alginate lyases. For instance, the activities of AlyPL17 towards sodium alginate, polyM and polyG are2.0, 1.71 and 3.97 times higher than the activities ofAlg17B(MacDonald et al., 2016). Moreover, the activities of AlgSH17 towards sodium alginate, polyM and polyG were 55.62 U/mg, 116.8U/mg, 30.28U/mg, respectively, which are 14.53, 7.73 and 26.50 times lower than that of AlyPL17(Yang et al., 2021). Accordingly, AlyPL17wasanexcellent tool for the degradation of alginate. In contrast, two truncated mutants (AlyPL17-N and AlyPL17-C) maintained only 3–5% of its initial activity. It indicates that both AlyPL17-N and AlyPL17-C domains were essential for maintaining activity. Furthermore, the km values of AlyPL17 with polyM, sodium alginate and polyG as substrates were 3.35 ± 0.62, 3.62 ± 0.23 and 4.89 ± 0.71mM, respectively. Thus, AlyPL17 was considered to prefer polyM block region in alginate polymer. Additionally, the kcat values of AlyPL17 towards polyM, sodium alginate and polyG were 39.42 ± 1.69, 38.30 ± 2.12 and 32.53 ± 0.88s− 1, respectively. It suggests that AlyPL17was a novel alginate lyase specific for polyM. Similarly, most alginate lyases from the PL17 family were polyM-preferred lyases, such as OalC17 from Cellulophaga sp.SY116 (Wang et al., 2014), AlgL17 from Microbulbifer sp. ALW1(Jiang et al., 2019), and Smlt2602 from Stenotrophomonas maltophilia K279a (MacDonald et al., 2016).
Table 1
substrate specificity and kinetics of AlyPL17
Substrate
|
Sodium alginate
|
PolyM
|
PolyG
|
Activity (U/mg)
|
808.53 ± 4.23
|
861.62 ± 3.53
|
802.62 ± 3.45
|
Km (mM)
|
3.62 ± 0.23
|
3.35 ± 0.62
|
4.89 ± 0.71
|
Vmax (µmol/s)
|
9.96 ± 0.95
|
10.24 ± 0.68
|
8.45 ± 1.02
|
kcat (s− 1)
|
38.30 ± 2.12
|
39.42 ± 1.69
|
32.53 ± 0.88
|
kcat/ Km(s− 1/mM)
|
10.58 ± 2.15
|
11.76 ± 2.36
|
6.65 ± 0.77
|
Biochemical Characterization of AlyPL17 and Its Truncated Mutants
For biochemical characterization of AlyPL17, AlyPL17-Nand AlyPL17-C, both AlyPL17 and its truncated mutants exhibited the highest activities at 45 oC (Fig. 4A) and pH 9.0 (Fig. 4D). As shown in Table 2, the optimal reaction conditions of alginate lyases from PL17 family are in the range of 30 to 50oC, and pH 6.0 to 8.0. For example, OalS17 (Wang et al., 2014) from Shewanella sp. Kz7 showed the highest activity at 50oC and pH 6.2, and TcAlg1 from Thalassotalea crassostreae exhibited the maximal activity at 40oC and pH 7.0 (Wang et al., 2018). Moreover, the optimal temperature and pH of Oalv17 were 40oC and 7.2(Li et al., 2020), respectively. In terms of temperature stability, AlyPL17 retains about 40%activityafter being incubated at 40oC for 1h (Fig. 4B), which was better than several PL17 family alginate lyases. For instance, OalC17 retains 20% of maximal activity after being incubated at 40oC for 1h (Li et al., 2018), and AlgSH17 could only retain stability at 20-25oC (Yang et al., 2021). Interestingly, the two truncated mutants exhibited excellent thermal stability, AlyPL17-N and AlyPL17-C could retain about 95% and 80% of its maximal activities after being incubated at 40oC for1 h (Fig. 4C). This phenomenon was also shown in truncated mutant Aly7B-CDII, which exhibited greater temperature stability than full-length enzyme Aly7B at 35oC(Hu et al., 2019). Similarly, the thermal ability of truncated AlgH-I and AlgH-II are 2.1 and 1.3-fold higher than full-length enzyme AlgH (Yan et al., 2019). Perhaps the truncated mutants have a more compact structure than full-length enzyme and thereby resists the effects of thermal degeneration (Hu et al., 2019). AlyPL17 reserves more than 60% of its maximal activities after being incubated at pH 6.0–9.0 for 24 h. Whereas, AlyPL17-N and AlyPL17-C maintained better stability at pH 9.0 (Fig. 4E). In further characterization, we introduced various metal ions to explore the effects on enzymatic activity. As shown in Fig. 4F, AlyPL17 was activated by Mg2+ and Mn2+. Moreover, AlyPL17-N could be activated by Na+. In addition, Co2+, Zn2+, Cu2+ and Fe3+ can inhibit the activities of AlyPL17 and its truncated mutants, as known for many other alginate lyases from PL17 Family.
Table 2 Characteristics of alginate lyases from PL17 family
Enzyme
|
Action mode
|
Optimal temperature/pH
|
Temperature/pH
stability
|
Substrate
preference
|
Source
|
Reference
|
|
AlyPL17
|
endo+exo
|
45oC/9.0
|
35oC, 60 min, remained 97%;
pH 6.0-9.0, 24h, remained 60%
|
polyM
|
Pedobacter hainanensis NJ-02
|
This study
|
|
Oal17A
|
exo
|
30oC/7.0
|
45oC, pH 6.0-9.0, remained> 80%
|
polyMG
|
Vibrio sp. W13
|
(Lyu et al., 2018)
|
|
OalV17
|
exo
|
40oC/7.2
|
40oC, 60 min, remained 74.5%;
pH 3.4-8.0, 12h, remained 70%
|
polyM
|
Vibrio sp. SY01
|
(Li et al., 2020)
|
|
OalB
|
exo
|
30oC/7.0
|
NA
|
polyMG
|
Vibrio splendidus12B01
|
(Jagtap et al., 2014)
|
|
OalC
|
exo
|
35oC/7.5
|
NA
|
polyM
|
Vibrio splendidus12B01
|
(Jagtap et al., 2014)
|
|
OAL
|
endo+exo
|
37oC/7.5
|
NA
|
polyM
|
StenotrophomonasmaltophiliaKJ-2
|
(Shin et al., 2015)
|
|
Alg17C
|
exo
|
40oC/6.0
|
NA
|
polyM, polyG
|
Saccharophagus degradans 2-40
|
(Park et al., 2014)
|
|
Alg17B
|
endo+exo
|
45oC/7.5-8.0
|
40oC, 60 min, remained 40%;
pH 6.5-8.0, 24h, remained 75%
|
Bi-
|
Vibriosp.BP-2
|
(Huang et al., 2019)
|
|
FlAlyB
|
exo
|
NA
|
NA
|
polyM
|
Flavobacterium sp. strain UMI-01
|
(Nishiyama et al., 2017)
|
|
TcAlg1
|
exo
|
40oC/7.0
|
40oC, 60 min, remain unchanged
|
polyM
|
Thalassotalea crassostreae
|
(Wang et al., 2018)
|
|
OalS17
|
exo
|
50oC/6.2
|
40oC, 60 min, remained 88%
pH 6.0-9.0, 6h, remained stable
|
polyMG
|
Shewanella sp. Kz7
|
(Wang et al., 2014)
|
|
AlgL
|
endo+exo
|
50oC/6.5
|
NA
|
polyM
|
Sphingomonas sp. MJ-3
|
(Jiang et al., 2019)
|
|
OalC17
|
exo
|
50oC/7.2
|
40oC, 60 min, remained 20%;
pH 6.0-8.0, 6h, remained 85%
|
polyM
|
Cellulophaga sp.SY116
|
(Li et al., 2018)
|
|
Smlt2602
|
exo
|
-/8.5
|
NA
|
polyM
|
Stenotrophomonas maltophilia K279a
|
(MacDonald et al., 2016)
|
|
AlgSH17
|
endo+exo
|
30oC/7.0
|
40oC, 60 min, remained less than 20%;
pH 6.0-8.0, 2h, remained 80%
|
polyM
|
Microbulbifer sp. SH- 1
|
(Yang et al., 2021)
|
Analysis of Action Pattern and Degradation Products of AlyPL17 and Its Truncated Mutants
Degradation products of AlyPL17 and its truncated mutants towards sodium alginate at various times (0–48 h) were analyzed by Fast protein liquid chromatography (FPLC). As shown in Fig. 5A, at preliminary stage of reaction, AlyPL17 could degrade alginate into dimers and monosaccharides. However, dimers were degraded into monosaccharides after incubation for 48 h. In accordance with the results above, AlyPL17 adopts a unique action mode that combines the endolytic action pattern and exolytic action pattern. ESI-MS was used to analyze the composition of the end products (Fig. 6A), one molecular ion peak at 175.02 m/z [ΔDP1-H]− was individually detected in the corresponding fraction. As shown in Table 2, although most alginate lyases from thePL17 family adopt exolytic mode to degrade alginate such asOalC17 from Cellulophaga sp.SY116 (Li et al., 2018)and FlAlyB from Flavobacterium sp. strain UMI-01(Nishiyama et al., 2017).This unique hybrid action pattern also has been adopted by several alginate lyases of PL17 family. For example, Alg17B from BP-2 (new species) could degrade alginate into alginate oligosaccharides with DP1-6 at preliminary stage of reaction, and then degrade alginate oligosaccharides into disaccharides and monosaccharides (Huang et al., 2019). Moreover, in the early stages of the reaction, OAL from StenotrophomonasmaltophiliaKJ-2 could produce alginate oligosaccharides with DP 1–4, and gradually degrade oligosaccharide into monosaccharide. Furthermore, AlgL from Sphingomonas sp. MJ-3 degrades alginate into alginate oligosaccharides with DP2-4 in endolytic action mode, and then it could degrade alginate oligosaccharides into monosaccharide in exolytic action mode (Jiang et al., 2019). Additionally, AlgSH17 from Microbulbifer sp. SH-1 also possesses both endolytic and exolytic activity (Yang et al., 2021). To explore the functions of the two domains, we analyzed the products distribution of AlyPL17-N and AlyPL17-C.As shown in Fig. 5B, AlyPL17-N exhibited a typical endolytic action pattern, which could degrade alginate into dimers, trimers, and tetramers. The final products had three molecular ion peaks at 268.79 m/z [DP3 + H2O-H]2−, 351.72 m/z [DP2-H]−,657.98 m/z [DP4 + 4H-3Na]− (Fig. 6B). In addition, AlyPL17-C also showed endolytic degradation mode towards sodium alginate(Fig. 5C), and the main products of AlyPL17-Cwere oligosaccharide with DP1-4(Fig. 6C).The final products had four molecular ion peaks at 160.84 m/z [Δ-H2O + 3H]2−, 268.79 m/z [DP3 + H2O-H]2−, 351.72 m/z [DP2-H]−, 657.98 m/z [DP4 + 4H-3Na]− (Fig. 6C). Similarly, the two truncated mutants of OAL adopt endolytic action mode to degrade alginate, and the main products of the mutants are DP1-6 (Shin et al., 2015). The hybrid activity of AlyPL17 perhaps is caused by the synergistic effects of AlyPL17-N and AlyPL17-C.
Synergistic Effect of AlyPL17 and AlyPL6
Although AlyPL17 exhibited relatively higher activities towards alginate than most of alginate lyases from PL17 family, it still showed lower catalysis efficiency than most endolytic alginate lyases. In our previous work, we cloned and characterized a novel endolytic alginate lyase AlyPL6 with high activity from Pedobacter hainanensis NJ-02 (Li et al., 2020). Since AlyPL17 has the ability to degrade alginate oligosaccharides into monosaccharide, it may prefer digesting the oligosaccharides with low DPs. Additionally, AlyPL17 and AlyPL6 were isolated from the same strain and have similar optimal reaction conditions. Last but not least, AlyPL17 and AlyPL6 have the complementary substrate specificity, AlyPL6 was polyMG preferred alginate lyase, and AlyPL17 exhibited the highest activities towards polyM. Therefore, AlyPL6and AlyPL17 may exhibit a synergistic degradation effect towards alginate. To investigate this possibility, the enzymatic assay was applied to evaluate the catalytic ability of AlyPL6 plus AlyPL17 (AlyPL6/AlyPL17 ratio: 0, 1:9, 2:8, 3:7, 4:6, and 5:5).
As shown in Supplementary Table S1, as the percentage of AlyPL6 goes up, the activities towards alginate have been improved obviously. When the ratio of AlyPL6/AlyPL17 reaches 4:6, the degradation activity towards alginate reached the maximum. Furthermore, in order to compare the monosaccharide yield of AlyPL6 plus AlyPL17 (AlyPL6/AlyPL17 ratio: 4:6) and AlyPL17 alone, FPLC was used for products analysis. As shown in Fig. 7A, a total of 10µg AlyPL6 plus AlyPL17 (4µg AlyPL6 and 6µg AlyPL17) could convert 10 mg of alginate into unsaturated monosaccharide with a yield of 21.4% after being incubated 10 min. In addition, the monosaccharides yield of AlyPL6 plus AlyPL17 (45%) was 1.96 times higher than AlyPL17 (23%) after being incubated at 45oC for 1h (Fig. 7B). Similarly, Li et al. established the method for the production of monosaccharide by combining endolytic alginate lyase AlySY08 and exolytic alginate lyases (OalC6 and OalC17) (Li et al., 2018). However, the activities and the temperature stabilities of OalC6 and OalC17 are weaker than AlyPL6 and AlyPL17 (Li et al., 2018). Accordingly, the combination of AlyPL6 and AlyPL17 is an efficient and stable tool for the production of biofuels from alginate.