Ecient Degradation of Alginate And Preparation of Unsaturated Monosaccharides By A Novel Alginate Lyase And Effects of Domain Truncation On Biochemical Characteristics, Degradation Patterns

Brown algae are considered promising crops for the production of sustainable biofuels. However, its commercial application has been limited by lack of ecient methods for converting alginate into fermentable sugars. Recently, exo-type alginate lyases have received extensive attention due to their excellent ability of conversion of alginate into 4-deoxy-L-erythro-5-hexoseulose uronate (DEH), a promising material for bioethanol production and biorenery systems. Herein, we cloned and characterized a novel alginate lyase AlyPL17 from Pedobacter hainanensis NJ-02. It possessed outstanding catalytic eciency towards polymannuronic acid (polyM), polyguluronic acid (polyG) and alginate sodium, with k cat of 39.42 + 1.9 s -1 , 32.53 + 0.88 s -1 , and 38.30 + 2.12 s -1 , respectively. In addition, AlyPL17 adopts a unique hybrid action mode to degrade alginate by the synergistic effect of two domains. Furthermore, the combination of AlyPL17 and AlyPL6 exhibited apparently synergistic effect for the preparation of unsaturated monosaccharides. Overall, the results show that AlyPL17 is a PL17 exo-type alginate lyase with high activity and a high conversion rate at low/moderate temperatures, which provides a useful enzymatic tool for the conversion of brown algae into biofuels and enhance our understanding of the function of modular domain of alginate lyase. high activity. Nevertheless, two truncated mutants AlyPL17-N and AlyPL17-C showed decreased activities, which indicated that both AlyPL17-N and AlyPL17-C domains are essential to maintain high activity of the full-length enzyme. AlyPL17 could degrade alginate into monosaccharide by a hybrid action mode that combined the endolytic action pattern and exolytic action pattern. However, AlyPL17-N and AlyPL17-Cexhibited an endolytic action pattern towards alginate. It suggested that AlyPL17-N and AlyPL17-C might show a synergistic effect, resulting in high exolytic activity of full-length enzyme AlyPL17. Last but not least, AlyPL6 and AlyPL17 exhibit excellent synergistic effects. When AlyPL6 and AlyPL17 were combined in a ratio of 4:6, the unsaturated monosaccharide conversion rate reached 45% in one hour. This work provides ecient enzymes for the development of biofuels from marine brown algae. Analysis of the Synergistic Effect of AlyPL17 and AlyPL6 To investigate the optimal synergistic degradation ratio, 20mL AlyPL6 plus AlyPL17 (0.2 mg/mL, AlyPL6/AlyPL17 ratio:0, 1:9, 2:8, 3:7, 4:6, and 5:5) were added to 180 mL alginate (5 mg/mL in 50 mM glycine-NaOH buffer pH 9.0) and incubated at 45 o C for 10 min. Ultraviolet absorption method was applied to measure the activity ofAlyPL6 plus AlyPL17. Furthermore, the composition of end products of optimal synergistic degradation ratio were analyzed by FPLC as previously reported (Li et al., 2020).


Abstract Background
Brown algae are considered promising crops for the production of sustainable biofuels. However, its commercial application has been limited by lack of e cient methods for converting alginate into fermentable sugars. Recently, exo-type alginate lyases have received extensive attention due to their excellent ability of conversion of alginate into 4-deoxy-L-erythro-5-hexoseulose uronate (DEH), a promising material for bioethanol production and biore nery systems.

Results
Herein, we cloned and characterized a novel alginate lyase AlyPL17 from Pedobacter hainanensis NJ-02. It possessed outstanding catalytic e ciency towards polymannuronic acid (polyM), polyguluronic acid (polyG) and alginate sodium, with k cat of 39.42 + 1.9 s -1 , 32.53 + 0.88 s -1 , and 38.30 + 2.12 s -1 , respectively. In addition, AlyPL17 adopts a unique hybrid action mode to degrade alginate by the synergistic effect of two domains. Furthermore, the combination of AlyPL17 and AlyPL6 exhibited apparently synergistic effect for the preparation of unsaturated monosaccharides.

Conclusion
Overall, the results show that AlyPL17 is a PL17 exo-type alginate lyase with high activity and a high conversion rate at low/moderate temperatures, which provides a useful enzymatic tool for the conversion of brown algae into biofuels and enhance our understanding of the function of modular domain of alginate lyase.

Background
With the global demand for renewable energy production increased markedly, methods for the production of bioethanol have been signi cantly improved in the past decade (Wargacki et al., 2012). Corn, sugarcane and several inedible lignocellulosic materials are preferable feedstocks (Takeda et al., 2011). However, it is di cult to convert the inedible lignocellulosic materials into unsaturated monosaccharides. In addition, using edible biomass could lead to bioethanol-food con icts. Last but not least, large-scale cultivation of biofuel crops may lead to a net increase in greenhouse gases (Takeda et al., 2011). Requiring no arable land, and fresh water resources, marine algae has been considered as the third-generation feedstocks (Takeda et al., 2011).
The most abundant sugars in brown alga are alginate, mannitol and glucan ( such as cellulose and laminarin)(Enquist-Newman et al., 2014). In recent years, many researchers have developed various strategies for fermenting these sugars into bioethanol (Wargacki et al., 2012). However, the utilization of alginate for bioethanol production is still a major bottleneck. Alginate, an acidic polysaccharide, consists of β-D-mannuronic acid (M) and α-L-guluronic acid (G) (Zhu & Yin, 2015). Several researchers have developed metabolic engineered microorganisms for fermenting alginate into ethanol. For example, Takeda et al. constructed a metabolically modi ed bacterium, Sphingomonas sp. A1, which could accumulate ethanol using alginate as the sole carbon source (Takeda et al., 2011). The metabolic pathway of alginate is as follows: rstly, endolytic alginate lyase could cleave the glycosidic bonds to create alginate oligosaccharides with various degrees of polymerization , while exolytic lyase could release unsaturated monosaccharides or disaccharides as products (Tøndervik et al., 2010). Unsaturated monosaccharides could be converted into4-deoxy-L-erythron-5-hexoseuloseuronate acid (DEH) through spontaneous conversion or KdgF catalysis (Hobbsa et al., 2017). Then DEH can be reduced to 2-keto-3-dexoxy-D-gluconate (KDG) before entering the Entner-Doudoroff (ED) pathway to produce two molecules of pyruvate, which may converted to two molecules of ethanol (Hobbsa et al., 2017) (Fig. 1). Thus, the degradation of alginate into unsaturated monosaccharides is an essential step for the preparation of bioethanol from alginate.
Various exolytic alginate lyases from several polysaccharide lyase families have been cloned and characterized in recent years. For instance, AlyGC (PL6 family) from Glaciecola chathamensis S18K6 T (Xu et al., 2017), VxAly7D (PL7 family) from Vibrio xiamenensis QY104 (Tang et al., 2020), Alg17c (PL17 family) from Saccharophagus degradans2-40  and Atu3025 (PL15 family) from Agrobacterium tumefaciens (Ochiai et al., 2010). Nevertheless, most exolytic alginate lyases have low activity, resulting in low unsaturated monosaccharide yield. In addition, almost all of the alginate metabolic bacteria contain two or three putative alginate lyases with different action mode and substrate speci city . However, complementary or synergistic effects of those alginate lyases have been rarely evaluated . Thus, evaluating synergistic effects of the alginate lyase from the same bacteria, and establishing an e cient method for the production of unsaturated monosaccharides are urgent problems to be solved.
In our previous work, we cloned and expressed a novel endolytic alginate lyase AlyPL6 from Pedobacterhainanensis NJ-02, which could degrade alginate into alginate oligosaccharides with high e ciency . Herein, we cloned and characterized a new PL17 family alginate lyase AlyPL17 from the same bacteria. The biochemical characterization and action pattern of AlyPL17were investigated. Additionally, we initially explore the mechanism of action mode of this enzyme through domain truncation. We hypothesized that AlyPL6 and AlyPL17 exhibit better synergistic effects, and can degrade alginate completely. This study is expected to provide e cient enzyme for the degradation of alginate and production of biofuels.

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, Lys 37 -Gln 265 ) and a heparinase II/III-like domain (designed as AlyPL17-C, Arg 357 -Phe 528 ). 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. 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% con dence. 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 Puri cation of AlyPL17 and Its Truncated Mutants
After heterologously expressed and puri ed, the recombinant AlyPL17, AlyPL17-N and AlyPL17-C were obtained and then analyzed by SDS-PAGE. In 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 k m 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 k cat 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 speci c for polyM. Similarly, most alginate lyases from the PL17 family were polyM-preferred lyases, such as OalC17 from Cellulophaga sp.SY116  For biochemical characterization of AlyPL17, AlyPL17-Nand AlyPL17-C, both AlyPL17 and its truncated mutants exhibited the highest activities at 45 o C (Fig. 4A) and pH 9.0 (Fig. 4D). As shown in Table 2 . 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 Mg 2+ and Mn 2+ . Moreover, AlyPL17-N could be activated by Na + . In addition, Co 2+ , Zn 2+ , Cu 2+ and Fe 3+ can inhibit the activities of AlyPL17 and its truncated mutants, as known for many other alginate lyases from PL17 Family. 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

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 e ciency 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 . 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 speci city, 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 45 o C 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) . However, the activities and the temperature stabilities of OalC6 and OalC17 are weaker than AlyPL6 and AlyPL17 . Accordingly, the combination of AlyPL6 and AlyPL17 is an e cient and stable tool for the production of biofuels from alginate.

Conclusion
In conclusion, a novel PL17 family AlyPL17 from Pedobacter hainanensis NJ-02 was cloned and expressed. Its biochemical properties and action pattern were clari ed. Furthermore, to investigate the function of the two domains, the N-terminal domain (AlyPL17-N) and Cterminal (AlyPL17-C) domain of AlyPL17 were subcloned and expressed, independently. AlyPL17 shared the highest identity of 45% with OAL and showed optimal activity at 45 o C and pH 9.0. Compared with other alginate lyases from the PL17 family, AlyPL17 has relatively high activity. Nevertheless, two truncated mutants AlyPL17-N and AlyPL17-C showed decreased activities, which indicated that both AlyPL17-N and AlyPL17-C domains are essential to maintain high activity of the full-length enzyme. AlyPL17 could degrade alginate into monosaccharide by a hybrid action mode that combined the endolytic action pattern and exolytic action pattern. However, AlyPL17-N and AlyPL17-Cexhibited an endolytic action pattern towards alginate. It suggested that AlyPL17-N and AlyPL17-C might show a synergistic effect, resulting in high exolytic activity of full-length enzyme AlyPL17. Last but not least, AlyPL6 and AlyPL17 exhibit excellent synergistic effects. When AlyPL6 and AlyPL17 were combined in a ratio of 4:6, the unsaturated monosaccharide conversion rate reached 45% in one hour. This work provides e cient enzymes for the development of biofuels from marine brown algae.

Materials And Methods
Materials and Strains.
Sodium alginate from Macrosystis pyrifera was purchased from Sigma-Aldrich (

Substrate Speci city and Enzymatic Kinetics
The activities of AlyPL17and its two truncated mutants were investigated using the ultraviolet absorption method described by Inoue

Biochemical Characterization of AlyPL17 and Its Truncated Mutants
To determine the optimal temperature and the thermal stability of AlyPL17, AlyPL17-N and AlyPL17-C, reactions were carried out at 30 o C-55 o C. Additionally, the thermally induced denaturation was also investigated by evaluating the residual activities after incubating at 40-45 o Cfor 0-60 min. The in uence of pH on enzymatic activities of these enzymes was studied by measuring the activities in presence of different buffers (50mM phosphate-citrate (pH 4.0-5.0), 50 mM NaH 2 PO 4 -Na 2 HPO 4 (pH 6.0-8.0), 50 mM Tris-HCl (pH 7.0-9.0) and 50 mM glycine-NaOH (pH 9.0-10.0)). Moreover, the pH stability was determined through assessing the residual activities after being incubated in different pH buffers at 4 o C for 24h. Furthermore, to investigate the effects of metal ions on enzymatic activities, puri ed enzymes were incubated with various metal ions (1mM) at 4 o C for 4 h. The reaction mixture without any metal ions was regarded as control (100% relative activity). In order to ensure the accuracy of the experiments, all experiments were performed with three replicates.

Action Pattern and Degradation Products Analysis
In order to explore the action pattern and degradation products of AlyPL17and its truncated mutants, 50 mL puri ed enzymes (10 mg) were incubated with 450 mL 0.5% sodium alginate at 40 o C for 0-48 h. The samples were taken after reaction for 1, 8, 24, 48h, respectively. Fast protein liquid chromatography equipped with superdex peptide 10/300 GE Column (GE Health) was applied to analyze the degradation products of AlyPL17 and its two truncated mutants as previously reported . In addition, the end products of AlyPL17 and its truncated mutants 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.
Ultraviolet absorption method was applied to measure the activity ofAlyPL6 plus AlyPL17. Furthermore, the composition of end products of optimal synergistic degradation ratio were analyzed by FPLC as previously reported .

Declarations
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Consent for publication
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Competing interests
The authors declare that they have no competing interests.   The pH stability of AlyPL17 and its two truncated mutants. (F) The in uence of metal ions of AlyPL17 and its two truncated mutants.

Supplementary Files
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