Elucidation of degradation pattern and immobilization of a novel alginate lyase for preparation of alginate oligosaccharides

Journal Pre-proof Elucidation of degradation pattern and immobilization of a novel alginate lyase for preparation of alginate oligosaccharides

Qian Li, Fu Hu, Mingyang Wang, Benwei Zhu, Fang Ni, Zhong Yao PII:

S0141-8130(19)37118-1

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.12.238

Reference:

BIOMAC 14266

To appear in:

International Journal of Biological Macromolecules

Received date:

3 September 2019

Revised date:

24 December 2019

Accepted date:

27 December 2019

Please cite this article as: Q. Li, F. Hu, M. Wang, et al., Elucidation of degradation pattern and immobilization of a novel alginate lyase for preparation of alginate oligosaccharides, International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/ j.ijbiomac.2019.12.238

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© 2018 Published by Elsevier.

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Elucidation of degradation pattern and immobilization of a novel alginate lyase for preparation of alginate oligosaccharides Qian Li1, Fu Hu1, Mingyang Wang2, Benwei Zhu1,, Fang Ni1, Zhong Yao1 1

College of Food Science and Light Industry, Nanjing Tech University, 30 Puzhu Rd,

2

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Nanjing 211816, P. R. China College of Computer Science and Technology, Nanjing Tech University, 30 Puzhu Rd,

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Nanjing 211816, P. R. China

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Emails for all authors: [email protected]; [email protected];

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[email protected]; [email protected]; [email protected];

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[email protected]; [email protected]



Corresponding author: [email protected];College of Food Science and Light Industry,

Nanjing Tech University, 30 Puzhu Rd, Nanjing211816, P R China 1

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Abstract Alginate lyases are important enzymes to prepare alginate oligosaccharides for industrial applications and elucidating the degradation pattern of alginate lyases is essential for expanding their applications. Herein, we cloned and expressed AlyPL6, a novel member of polysaccharide lyase family-6 (PL6) with high activity from

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Pedobacter hainanensis NJ-02. It was found that AlyPL6 could recognize tetrasaccharide as the minimal substrate to release oligosaccharides with low degrees

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of polymerization (DPs). As a result, it could be speculated that the cleavage site of

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substrate is located between -1 and +1 subsites. For further application, AlyPL6 was

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then immobilized onto mesoporous titanium oxide particles (MTOPs) with more than

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55.4% of maximal activity retained at 45°C after it was reused for 10 times. The

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research provided extended insights into the substrate recognition and degradation

lyases a lot.

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pattern of PL 6 alginate lyases, which may benefit the further application of alginate

Keywords Alginate lyase; Polysaccharide lyase 6 family; Characterization; Immobilization; Mesoporous titanium oxide particles; Degradation pattern.

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1. Introduction Alginate is a linear acidic polysaccharide that acts as the major structural component of the cell wall of brown algae (Gacesa, 1992). It consists of randomly arranged -L-guluronate (G) and -D-mannuronate (M) (Lee & Mooney, 2012). The alginate possesses favorable physicochemical properties and has been widely used in food industrial fields. For example, as the food stabilizer and thickener, alginate can

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improve the quantity and stability of the bread and ice cream (Turquois & Gloria, 2000). However, the applications of alginate have been greatly limited due to its low

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bioavailability and high molecular weight. Alginate could be degraded into alginate

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oligosaccharides. Alginate oligosaccharides have various advantages such as

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prominent solubility, bioavailability and have been widely used as food additives and

lP

protective agents of perishable food. For example, it showed positive effects on the firmness, springiness and chewiness of shrimp meat (Ma, Zhang, Deng, & Xie, 2015).

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In addition, as a probiotic factor, alginate oligosaccharides can promote the proliferation of bifidobacterium and regulate intestinal micro-ecological balance

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(Wang, Han, Hu, Li, & Yu, 2006). As a result, preparing the alginate oligosaccharides has been the focus in marine food industry (Falkeborg, Cheong, Gianfico, Sztukiel, Kristensen, Glasius, et al., 2014). As a member of polysaccharide lyase family (PL), alginate lyases could degrade alginate to unsaturated alginate oligosaccharides with double bonds at the non-reducing ends by β-elimination mechanism (Wong, And, & Schiller, 2000). The preparation of alginate oligosaccharides using alginate lyase has many advantages such as high efficiency and mild degrading conditions. Accordingly, discovering and obtaining new alginate lyases with excellent properties have drawn increasing attentions in recent years (B. Zhu, Chen, Yin, Du, & Ning, 2016). So far, alginate 3

Journal Pre-proof lyases are classified into 12 polysaccharide lyase families (PLs), PL5, PL6, PL7, PL14, PL15, PL17, PL18, PL31, PL32, PL34, PL36 and PL39, according to the CAZy database. Various enzymes belong to different polysaccharide lyase families have been characterized (B. Zhu & Yin, 2015). As for PL 6 family, there are several enzymes have been identified and characterized such as TsAly6A from Thalassomonas sp. LD5, OalS6 from Shewanella sp. Kz7, AlyMG from Stenotrophomas maltophilia KJ-2, AlyGC of Glaciecola chathamensis S18K6T.

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Unfortunately, most of these alginate lyases had low activity and poor thermal

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& Ojima, 2016; B. Zhu, Ni, Sun, & Yao, 2017).

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stability, which greatly limited their industrial applications (Inoue, Anraku, Nakagawa,

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In our previous work, we have cloned and characterized two carrageenases from

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Pedobacter hainanensis NJ-02, which can hydrolyze carrageenan into even-numbered oligosaccharides (Benwei Zhu, Ni, Ning, Yao, & Du, 2017; B. W. Zhu, Xiong, Ni,

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Sun, & Yao, 2018). Moreover, it exhibited excellent degrading ability not only to

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carrageenan but also alginate. Herein, we successfully cloned a novel PL6 alginate lyase, AlyPL6, from Pedobacter hainanensis NJ-02. The biochemical characteristics and degradation pattern of AlyPL6 towards different substrates were investigated. In addition, AlyPL6 was further immobilized onto mesoporous titanium oxide particles (MTOPs) to improve thermal stability. The research could definitely provide extended insights into the substrate recognition and degradation pattern of PL 6 alginate lyases. 2. Materials and Methods 2.1 Materials and strains Sodium alginate was purchased from Sigma–Aldrich (M/G ratio 77/23 isolated from Macrosystis pyrifera, viscosity ≥2000 Cp, St. Louis, MO, USA). PolyG and polyM

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with average DPs of 39 (purity: about 95%; M/G ratio: 97/3 and 3/97; average molecular weight: 7200 Da) were purchased from Qingdao BZ Oligo Biotech Co., Ltd (Qingdao, China). OligoM with DP2-8 was purchased from Qingdao BZ Oligo Biotech Co., Ltd (Qingdao, China). Marine bacterium Pedobacter hainanensis NJ-02 was previously isolated from East China Sea and conserved in our laboratory (Benwei

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Zhu, Ni, Ning, Yao, & Du, 2017). 2.2 Sequence analysis.

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The alignment of protein sequences of AlyPL6 and other enzymes was

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performed with Vector-NTI (Life Technologies, Grand Island, NY). The HMM Pfam

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application of the InterProScan 4 was used to predict the conserved structural domains

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of AlyPL6. According to the protein sequences of PL6 family enzymes, the

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phylogenetic tree was constructed through Molecular Evolutionary Genetics Analysis (MEGA) Program version 6.0. Homology/analogY Recognition Engine V 2.0 was

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used to homology modeling.

2.3 Expression and purification of AlyPL6 According to the gene sequence that encoded putative alginate lyase (NJ-02_GM000789) of P. hainanensis NJ-02, the primers for cloning AlyPL6 were designed as described in Table S1 (Supplementary information). The pET-21a (+) plasmid was used as an expression vector to ligate the gene of AlyPL6, and then the recombinant plasmid was introduced into E. coli BL21 (DE3). The recombinant strain was cultured at 37°C up to an OD600 of 0.4–0.6 and the expression of recombinant enzyme was induced by the addition of 0.1 mM IPTG at 5

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20°C for 30 h. Afterwards, AlyPL6 was purified by Ni-NTA Sepharose column (GE Healthcare, Uppsala, Sweden), and it was analyzed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as previously described (Benwei Zhu, Ni, Ning, Yao, & Du, 2017). Additionally, the protein concentration was determined by protein quantitative analysis kit (Beyotime Institute of Biotechnology,

2.4 Substrate specificity and enzymatic kinetics

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Nantong, China).

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The purified AlyPL6 (3.6 ng in 30 L) was incubated with 270L 0.2%

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substrates (sodium alginate, polyM and polyG dissolved in glycine-NaOH buffer pH

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10.0) at 45°C for 5 min followed by incubating in boiling water for 10 min to

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terminate the reaction. One unit of enzymatic activity was defined as the amounts of

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enzyme required to increase absorbance at 235 nm (extinction coefficient: 6150 M-1.cm-1) by 0.1 per min.

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The substrate specificity of AlyPL6 was also characterized by determining the activities towards three different substrates (0.2% of polyM, 0.2% of sodium alginate and 0.2% of polyG). Various concentrations (0.2–6.0 mg/mL) of three substrates (alginate, polyM, PolyG) were used to investigate the kinetic parameters of AlyPL6 towards these substrates. The concentrations of the product and the reaction velocity (v) were calculated as previously described (Benwei Zhu, Ni, Ning, Yao, & Du, 2017). Based on hyperbolic regression analysis, Km and Vmax values were calculated (Studnicka, 1987). Then, the turnover number (kcat) of AlyPL6 was calculated by the ratio of Vmax versus enzyme concentration ([E]). 6

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2.5 Biochemical characterization of AlyPL6 In order to determine the optimal pH for AlyPL6 activity, the purified enzyme was mixed with the substrate in buffers with different pHs (4.0-12.0) and the activity was measured under the standard conditions described as follows. Detailly, the purified enzyme was mixed with the substrate in buffers with different pHs , which were 50 mM phosphate-citrate (pH 4.0–5.0), 50 mM NaH2PO4-Na2HPO4 (pH 6.0–

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8.0), 50 mM Tris–HCl (pH 7.0–9.0), and 50 mM glycine-NaOH (pH 9.0–10.0)at 45oC

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for 30min.

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Furthermore, the pH stability was evaluated according to the residual activity

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after being incubated in buffers with diverse pHs (4.0–12.0) for 12h. In order to

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times at the same conditions.

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improve the credibility of the experimental data, each experiment performed three

AlyPL6 reaction was carried out at 30-60oC to investigated its optimal

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temperature and thermal stability. In addition, the thermally-induced denaturation was also inspected by testing the residual activity after incubating the enzyme at 35-50°C for 0-60 min to assess its thermal stability. Different metal ions (1 mM) were used to explore the effects of metal ions on the activity of AlyPL6 by incubating AlyPL6 at 4°C for 12 h. In addition, the activity of reaction mixture without any metal ion was regarded as control (100% relative activity). 2.6 Action pattern and degradation products analysis In order to investigate the action pattern of AlyPL6, the products were separated using FPLC with a Superdex peptide 10/300 GE Colum (GE Health) and monitored at 7

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the wavelength of 235 nm. The mixture was eluted with 0.2M NH4HCO3 at flow rate of 0.5mL/min. In addition, the composition of the degrading products was analyzed by ESI-MS as previously reported (Benwei Zhu, et al., 2017). To identify the size of the substrate-binding subsite of catalytic tunnel in AlyPL6, 10 μL reaction mixture (including 2 ng AlyPL6 and 10 mg/mL of oligomannuroates

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(oligoM) with different DPs of 2-8) were incubated at 30°C for 48 h. ESI-MS was

previously described (Benwei Zhu, et al., 2017).

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2.7 Molecular modeling

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used to analyze the degradation products in a negative-ion mode with the settings as

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The online software Protein Homology/analogY Recognition Engine V (PHYRE)

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2.0 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) was applied to

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establish the three-dimensional structure of AlyPL6 according to the structure of alginate lyase AlyGC from Glaciecola chathamensisS18K6T (PDB ID: 5GKD) with

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sequence identity of 36%.

2.8 Immobilization of AlyPL6 and preparation of alginate oligosaccharides with immobilized AlyPL6.

Exactly 100 mg of mesoporous titanium oxide particles (MTOPs) were incubated with 1 mL of AlyPL6 (1.2 mg/mL) to immobilize AlyPL6 on MTOPs by stirring at 200 rpm for 3 h at 4 °C. The precipitated particles were then isolated by centrifugation at 8000 rpm for 10 min and washed five times with 50 mM Tris-HCl buffer (pH 8.0). The immobilization rate was determined by measuring the protein concentration of AlyPL6 before and after the immobilization. In addition, the yield of activity of 8

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immobilization was investigated through measuring the specific enzyme activity before and after the immobilization. The immobilized AlyPL6 (AlyPL6-MTOPs) was stored at 4 °C for further use. The thermal stability of AlyPL6-MTOPs was assayed by measuring the residual activity of free enzyme and immobilized enzyme after incubation for 60 min at various temperatures (35–60 °C). Additionally, the reusability

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of immobilized enzyme was determined by measuring the residual activity after repeat reaction 10 cycles.

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3. Results and discussion

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3.1 Sequence analysis of the alginate lyase gene

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As shown in Figure 1, the open reading frame (ORF) of AlyPL6 consists of 1359

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bp encoded a putative alginate lyase composed of 452 amino acids with a theoretical

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molecular weight of 49.74 kDa. Based on the results of the conserved domain analysis, AlyPL6 comprises only an N-terminal catalytic domain of 345 amino acids

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(Lys53-Asn397). To the best of our knowledge, all alginate lyases of PL6 family except AlyF isolated from Vibrio sp.OU02 (Lyu, et al., 2018) possess two structural domains such as AlyGC from Glaciecola chathamensis S18K6T (Xu, Dong, Wang, Cao, Li, Li, et al., 2017) and FsAlyPL6 from Flammeovirga sp. NJ-04 (Q. Li, Hu, Zhu, Sun, & Yao, 2019). AlyPL6 had the highest identity of 71% with AlyP from Pseudomonas sp. OS-ALG-9 (GenBank accession no.BAA01182.1) (Kraiwattanapong, Motomura, Ooi, & Kinoshita, 1999). According to the alignment of protein sequence, AlyPL6 contains the conserved regions such as “NG(G/A)E”, “(I/V)KS”, and “R(H/S)GN” (FigureS2), 9

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which contribute to the substrate binding and catalytic activity (Xu, et al., 2017). To further identify the subfamily of AlyPL6, the phylogenetic tree was constructed and it was found that AlyPL6 clusters with representative enzymes of subfamily 1 (Figure2), thus AlyPL6 is considered as a new member of the subfamily 1.

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3.2 Cloning, expression and substrate specificity of AlyPL6 After heterologously expressed, the purified AlyPL6 was obtained through

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Ni-NTA Sepharose affinity chromatography and then analyzed by SDS-PAGE. As

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shown in Figure 3, a clear band (about 50 kDa) represented the molecular mass of

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AlyPL6 can be observed, which is consistent with the predicted molecular mass of

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49.74 kDa. The molecular mass (Mr) of other PL6 alginate lyases varied from 47.8

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kDa to 107 kDa. KJ-2 from Stenotrophomas maltophilia KJ-2 possessed the smallest Mw of 47.8 kDa(Su, Choi, & Lee, 2012), while Rmar1386 from Rhodothermus

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marinus DSM 4252 had the biggest Mw of 107 kDa among PL6 enzymes (Mathieu, Henrissat, Labre, Skjåk-Bræk, & Helbert, 2016). Afterwards, in order to determine the substrate specificity of AlyPL6, three kinds of substrates (sodium alginate, polyM and polyG) were used for activity assay. As shown in Table 1, the recombinant AlyPL6 showed higher activity towards sodium alginate (1319.7 U/mg) than that to polyM (1052.1 U/mg) and polyG (1310.4 U/mg). Therefore, AlyPL6 is suggested to be a novel alginate lyase specific for polyMG just like other PL6 enzymes with an exception of Pedsa0631 which preferred polyG to polyMG (Mathieu, Henrissat, Labre, Skjåk-Bræk, & Helbert, 2016). In addition, 10

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Patl_3640

from

Pseudoalteromonas

atlantica

T6c

and

Pedsa_0631

from

Pseudopedobacter saltans DSM 12145 preferred polyG and polyMG block, which is different from other enzymes of PL6 family (Mathieu, Henrissat, Labre, Skjåk-Bræk, & Helbert, 2016). According to the hyperbolic regression analysis, the kinetic values (Km and Vmax)

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of AlyPL6 towards polyM, sodium alginate and polyG were calculated as shown in Table 1. The Km values of AlyPL6 with sodium alginate, polyG and polyM as

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substrates were 6.17 mM, 9.18 mM and 9.57 mM, respectively. Thus, AlyPL6 was

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considered to prefer polyMG block region in alginate polymer. The kcat values of

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AlyPL6 towards sodium alginate, polyM and polyG were 38.45 s-1, 18.60 s-1, and

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32.45 s-1, respectively. It suggested that AlyPL6 exhibited the higher catalytic efficiency towards MG-block than that towards G-block and M-block.

sodium alginate

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Substrate

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Table 1Substrate specificity and kinetics of AlyPL6 polyM

polyG

Activity(U/mg)

1319.7

1052.1

1310.4

Km (mM)

6.17

9.57

9.18

Vmax (mol/s)

12.42

6.01

10.48

kcat (s-1)

38.45

18.60

32.45

kcat/Km (s-1/mM)

6.23

1.94

3.53

3.3 Biochemical characterization of AlyPL6 AlyPL6 showed maximal activity at 45°C and could retain about 50% of maximal activity after being incubated at 40°C for 60 min (Figure 4B). Compared

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with AlyPL6, the other enzymes of PL6 family exhibited their maximal activity at lower temperatures. For instance, TsAly6A from Thalassomonas sp. LD5 showed maximal activity at 35°C and could retain 80% of maximal activity below 30°C (Shan, Zhelun, Shangyong, Hang, Luyao, Yulong, et al., 2018). OalS6 from Shewanella sp. Kz7 (S. Li, Wang, Han, Gong, & Yu, 2015) and AlyMG from Stenotrophomas

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maltophilia KJ-2 (Su, Choi, & Lee, 2012) showed their maximal activities at 40°C and maintained 80% of maximal activity below 40°C. AlyPL6 showed maximal

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activity at pH10.0 (Figure 4C). Furthermore, it could reserve more than half of its

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maximum activity after being incubated at pH 8.0–11.0 for 24 h (Figure4D). Thus, the

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enzyme was mostly stable at pH 10.0. Accordingly, AlyPL6 was an alkaline-stable

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lyase, while other enzymes usually showed the optimal activity at pH 7.2 or 8.0. For

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instance, OalS6 from Shewanella sp. Kz7 had an optimal pH of 7.2 (S. Li, Wang, Han, Gong, & Yu, 2015), while TsAly6A from Thalassomonas sp. LD5 (Shan, et al., 2018)

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and AlyMG from Stenotrophomas maltophilia KJ-2 (Su, Choi, & Lee, 2012) exhibited maximal activity at pH 8.0. In addition, after being incubated at 35°C with 50 mM glycine-NaOH (pH 10.0) for 50 min, AlyPL6 only lost approximately 10% activity and lost most of its activity with increased temperature (Figure 4B). These excellent characteristics indicated that AlyPL6 possessed great potential in applications of food industry. The effect of metal ions on enzyme activity was shown in Table 2, the activity of AlyPL6 can be activated by Na+ and inhibited by some divalent ions such as Mn2+, Zn2+, Cu2+ and Co2+. OalS6 from Shewanella sp. Kz7 and AlyMG from 12

Journal Pre-proof Stenotrophomas maltophilia KJ-2 can be both activated by K+, Na+, and Ca2+, while the activity of TsAlyA6 from Thalassomonas sp. LD5 cannot be obviously affected by K+ (Shan, et al., 2018). Table 2. The effects of metal ions on activity of AlyPL6 Reagent Relative Activity (%) 100.00 ± 2.97

K+

98.15 ± 2.23

Na+

102.35± 1.08

Ca2+

97.64 ± 1.12

Mg2+

91.19 ± 2.78

2+

39.73 ± 1.32

2+

11.68 ± 3.57

Cu2+

46.63 ± 1.20

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55.28 ± 3.93

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Ni2+

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Zn

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Co

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Control

5.02 ± 0.60

Fe3+

80.94 ± 1.21

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Mn2+

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3.4 Action pattern analysis and substrate docking of AlyPL6 The degradation products of AlyPL6 towards sodium alginate, polyG and polyM at different times (0-48 h) were also analyzed (Figure 5). At the preliminary stage of degradation, diverse sizes of alginate oligosaccharides (DPs 2-6) could be detected. However, a large proportion of substrates were degraded into dimers, trimers and tetramer after incubation for 48 h. Interestingly, monosaccharides appeared in the final stage of the degradation. In accordance with the results above, AlyPL6 can degrade the substrates in a unique manner. ESI-MS was used to analyze the composition of the degradation products (Figure 6). It can be observed that oligomers with DPs of 2–5 were released 13

Journal Pre-proof with alginate, polyM and polyG as substrates (Figure 6A, 6B and 6C). In line with the FPLC results, the peaks representing monosaccharide could be detected in the ESI-MS spectra. The result indicated thatAlyPL6 may be a potential tool to prepare alginate oligosaccharides with lower DPs. Several action patterns and the distribution of end products could be found in PL6 subfamily 1. Some of endo-type alginate lyase in subfamily 1, such as AlyMG from Stenotrophomas maltophilia KJ-2 which mainly produced oligosaccharides with DPs of 2–4 in an endolytic manner (Su, Choi, & Lee,

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2012) and the TsAly6A of Thalassomonas sp. LD5 generated dimeric and trimeric

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products (Shan, et al., 2018). However, some PL6 subfamily 1 enzymes could produce

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monosaccharides in an exo-type manner, such as AlyGC of Glaciecola chathamensis

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S18K6T (Xu, et al., 2017), OalS6 of Shewanella sp. Kz7 (S. Li, Wang, Han, Gong, & Yu, 2015). In addition, some of alginate lyases of PL6 subfamily 1 adopted an

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endo-type action pattern when substrates are polyM or polyMG and adopt exo-type

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action when substrates used as polyG. For example, Rmar_1165 of Rhodothermus marinus DSM 4252, Nonul_2381 of Nonlabens ulvanivorans PLR, Patl_3659 of

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Pseudoalteromonas atlantica T6c, Celly_0294 of Cellulophaga lytica DSM 7489, MASE_04135 of and Alteromonas macleodii ATCC 27126 (Mathieu, Henrissat, Labre, Skjåk-Bræk, & Helbert, 2016). However, AlyPL6 adopts the endo-type action pattern towards alginate, polyM and polyG. Additionally, this is the first time to report the endo-type alginate lyase of PL6 subfamily 1could produce monosaccharide during the hydrolytic procedure. Members of PL6 subfamily 2 and PL6 subfamily 3 could degrade sodium alginate into disaccharide and tetrasaccharide in an endolytic manner, such as Phep_3223 of Pedobacter heparinus DSM 2366, Rmar_1386 of Rhodothermus marinus DSM 4252, Sde_0034 of Saccharophagus degradans 2-40

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Journal Pre-proof and SVEN_0074 of Streptomyces venezuelae ATCC 10712 (Mathieu, Henrissat, Labre, Skjåk-Bræk, & Helbert, 2016). The substrate binding subsites of AlyPL6 were determined firstly. With the substrates concentration increased and the reaction time prolonged, the disaccharide and trisaccharide cannot be degraded by AlyPL6 (Data not shown), which indicated the tetrasaccharide was the minimal substrate that can be degraded by AlyPL6 to

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monosaccharide, disaccharide (dimannuronate) and trisaccharide (trimannuronate) as

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product (Figure7A). As a result, it could be speculated that the catalytic cavity of

(pentamannuronate),

hexasaccharide

(hexamannuronate),

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pentasaccharide

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AlyPL6 contains at least four subsites. The final products of AlyPL6 towards

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heptasaccharide (heptamannuronate) and octasaccharide (octamannuronate) were oligosaccharides with DPs of 1-4 (Figure7B-E). It is presumed that AlyPL6 can

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degrade tetrasaccharide into monosaccharide, disaccharide and trisaccharide through

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cleaving the first glycosidic bond from the non-reducing terminus at the site between subsities -1 and +1. While for pentasaccharide, hexasaccharide, heptasaccharide, and octasaccharide, they can be degraded into oligosaccharide with DPs (1-4) by cleaving the similar site. Based on the homologues structure of alginate lyase AlyGC from Glaciecola chathamensisS18K6T (PDB ID: 5GKD), the three-dimensional model of AlyPL6 was constructed using PHYRE2 with the sequence similarity of 36%. Despite the low sequence similarity between AlyPL6 and AlyGC, the protein model was successfully constructed with 100% confidence since they share the same folding pattern of right-handed -helix fold (Xu, et al., 2017). As shown in Figure 8A, the overall 15

Journal Pre-proof structure of AlyPL6 was predicted to fold into a “tower-like” structure with ten right-handed β helixes. Compared with the “twin tower-like” structure of AlyGC, AlyPL6 shared similar -helical structure with the N-terminal domain (Module A) of AlyGC, but lacked the C-terminal domain (Module B) of AlyGC (Figure 8B). Based on the analysis of the sequence alignment and protein-substrate interactions, the key residues for substrate recognition were identified. As indicated in Figure 8C, the residues K248 and R269 are highly conserved and involved in the interaction of the

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3.5 Characterization of immobilized AlyPL6

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protein and substrates in subsites −1and +1, respectively.

Thermal stability and reusability are important for enzymes in industrial

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applications. AlyPL6 was successfully immobilized onto the MTOPs with

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immobilization rate of 71.75% and the yield of immobilized enzyme activity was

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64.9%. As shown in Figure 9A, the free and immobilized AlyPL6 could retain 9.6% and 83.7% of the initial activity after heating at 45°C for 1 h, respectively. The results

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indicated that the thermal stability of AlyPL6 have been improved markedly after immobilization onto the MTOPs. Besides the thermal stability, the reusability is another important index to evaluate the application value of the immobilized enzyme. The reusability of immobilized AlyPL6 was shown in Figure 9B, the immobilized AlyPL6 exhibited a significantly higher reusability than free AlyPL6. The AlyPL6 immobilized on MTOPs retained 74.9% and 54.4% of the initial activity after 5 and 10 cycles. The reason for the excellent performance of immobilized AlyPL6 is that MTOPs can protect the structure and active sites of the enzyme. Meanwhile, compared with free AlyPL6, immobilized AlyPL6 presented an outstanding thermal stability and 16

Journal Pre-proof reusability. These results indicated that immobilization on MTOPs can significantly increase the stability and prevent leaching of the immobilized AlyPL6. 4. Conclusions It is essential to understand the action mode and products distribution of alginate lyases for further utilization in preparation of oligosaccharides. Herein, we firstly characterized and elucidated the unique degradation pattern of a novel alginate lyase

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of PL 6 family. In addition, in order to expand its industrial applications, we

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immobilized the enzyme onto MTOPs to improve the thermal stability and reusability

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of AlyPL6. This work would definitely provide the insight into action mode and

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catalytic mechanism of PL6 family alginate lyases. The last but not the least, it would

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greatly enhance the applications of the enzyme in food and agricultural field. Declaration of interests

Acknowledgment

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The authors declare that they have no known competing interests.

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The work was supported by the National Natural Science Foundation of China (Grant Nos. 31601410, 21776137). References

Falkeborg, M., Cheong, L. Z., Gianfico, C., Sztukiel, K. M., Kristensen, K., Glasius, M., Xu, X., & Guo, Z. (2014). Alginate oligosaccharides: enzymatic preparation and antioxidant property evaluation. Food Chem, 164, 185-194. Gacesa, P. (1992). Enzymic degradation of alginates. Int J Biochem, 24(4), 545-552. Inoue, A., Anraku, M., Nakagawa, S., & Ojima, T. (2016). Discovery of a Novel Alginate Lyase from Nitratiruptor sp. SB155-2 Thriving at Deep-sea Hydrothermal Vents and Identification of the Residues Responsible for Its Heat Stability. J Biol Chem, 291(30), 15551-15563. 17

Journal Pre-proof Kraiwattanapong, J., Motomura, K., Ooi, T., & Kinoshita, S. (1999). Characterization of alginate lyase (ALYII) from Pseudomonas sp. OS-ALG-9 expressed in recombinant Escherichia coli. World Journal of Microbiology & Biotechnology, 15(1), 105-109. Lee, K. Y., & Mooney, D. J. (2012). Alginate: Properties and biomedical applications. Progress In Polymer Science, 37(1), 106-126. Li, Q., Hu, F., Zhu, B., Sun, Y., & Yao, Z. (2019). Biochemical Characterization and Elucidation of Action Pattern of a Novel Polysaccharide Lyase 6 Family Alginate

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Lyase from Marine Bacterium Flammeovirga sp. NJ-04.Marine Drugs,17(6), 323.

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Li, S., Wang, L., Han, F., Gong, Q., & Yu, W. (2015). Cloning and characterization of

Kz7. Journal of Biochemistry, 159(1), 77.

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the first polysaccharide lyase family 6 oligoalginate lyase from marine Shewanella sp.

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Ma, L. K., Zhang, B., Deng, S. G., & Xie, C. (2015). Comparison of the

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Cryoprotective Effects of Trehalose, Alginate, and Its Oligosaccharides on Peeled Shrimp (Litopenaeus vannamei) During Frozen Storage. Journal Of Food Science,

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80(3), C540-C546.

Mathieu, S., Henrissat, B., Labre, F., Skjåk-Bræk, G., & Helbert, W. (2016).

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Functional Exploration of the Polysaccharide Lyase Family PL6. Plos One, 11(7),

Shan, G., Zhelun, Z., Shangyong, L., Hang, S., Luyao, T., Yulong, T., Wengong, Y., & Feng, H. (2018). Characterization of a new endo-type polysaccharide lyase (PL) family 6 alginate lyase with cold-adapted and metal ions-resisted property. International Journal of Biological Macromolecules, 120, 729-735. Studnicka, G. M. (1987). Hyperbolic regression analysis for kinetics, electrophoresis, ELISA, RIA, Bradford, Lowry, and other applications. Computer Applications in the Biosciences Cabios, 3(1), 9-16. Su, I. L., Choi, S. H., & Lee, E. Y. (2012). Molecular cloning, purification, and characterization of a novel polyMG-specific alginate lyase responsible for alginate MG block degradation in Stenotrophomas maltophilia KJ-2. Applied Microbiology & Biotechnology, 95(6), 1643-1653. 18

Journal Pre-proof Turquois, T., & Gloria, H. (2000). Determination of the absolute molecular weight averages and molecular weight distributions of alginates used as ice cream stabilizers by using multiangle laser light scattering measurements. Journal Of Agricultural And Food Chemistry, 48(11), 5455-5458. Wang, Y., Han, F., Hu, B., Li, J. B., & Yu, W. G. (2006). In vivo prebiotic properties of alginate oligosaccharides prepared through enzymatic hydrolysis of alginate. Nutrition Research, 26(11), 597-603. Wong, T. Y., And, L., & Schiller, N. L. (2000). Alginate Lyase: Review of Major

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Sources and Enzyme Characteristics, Structure-Function Analysis, Biological Roles,

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and Applications. Annual Review of Microbiology, 54(1), 289-340. Xu, F., Dong, F., Wang, P., Cao, H. Y., Li, C. Y., Li, P. Y., Pang, X. H., Zhang, Y. Z., &

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Chen, X. L. (2017). Novel Molecular Insights into the Catalytic Mechanism of Marine

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Bacterial Alginate Lyase AlyGC from Polysaccharide Lyase Family 6. Journal Of

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Biological Chemistry, 292(11), 4457-4468.

Zhu, B., Chen, M., Yin, H., Du, Y., & Ning, L. (2016). Enzymatic Hydrolysis of

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Zhu, B., Ni, F., Ning, L., Yao, Z., & Du, Y. (2017). Cloning and biochemical characterization of a novel κ-carrageenase from newly isolated marine bacterium Pedobacter hainanensis NJ-02. International Journal of Biological Macromolecules. Zhu, B., Ni, F., Sun, Y., & Yao, Z. (2017). Expression and characterization of a new heat-stable endo-type alginate lyase from deep-sea bacterium Flammeovirga sp. NJ-04. Extremophiles, 21(6), 1027-1036. Zhu, B., & Yin, H. (2015). Alginate lyase: Review of major sources and classification, properties, structure-function analysis and applications. Bioengineered Bugs, 6(3), 125-131. Zhu, B. W., Xiong, Q., Ni, F., Sun, Y., & Yao, Z. (2018). High-level expression and characterization of a new κ -carrageenase from marine bacterium Pedobacter hainanensis NJ-02. Letters in Applied Microbiology, 66(5). 19

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Figures captions Fig. 1 Multiple amino acid sequence alignment of AlyPL6 and other alginate lyases of PL 6 family: Phep_3223 (ACU05418) from Pedobacter heparinus DSM 2366, SVEN_0074 (CCA53362.1) from Streptomyces venezuelae ATCC 10712, Pedsa_3628 (ADY54157.1) from Pseudopedobacter saltans DSM 12145, AlyMG (AFC88009.1)

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from Stenotrophomonas maltophilia KJ-2, AlyP (BAA01182.1) from Pseudomonas sp. OS-ALG-9. Identical and similar amino acid residues among the alginate lyases are

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shaded in yellow. The locations of three conserved regions are marked with boxes.

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Residues responsible for enzymatic activity and catalysis are marked with stars and

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dots, respectively.

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Fig.2 Phylogenetic tree of AlyPL6 and other alginate lyases of PL 6 family based on amino acid sequence comparison. The species names are indicated along with accession numbers of corresponding alginate lyase sequences. Bootstrap values of 1000 trials are presented in the branching points. The alginate lyases of subfamily 1, 2

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and 3 are marked with stars, dots and triangles, respectively.

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Fig. 3 SDS-PAGE analysis of purified AlyPL6. Lane M protein: restrained marker

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(Thermo Scientific, USA); lane 1: purified AlyPL6.

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Fig. 4 Biochemical characterization of AlyPL6. (A) The temperature dependence and thermal stability of AlyPL6. (B) The thermal-induced denaturation of AlyPL6. (C)

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The optimal pH of AlyPL6. (D)The pH stability of AlyPL6.

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Fig. 5 FPLC analysis of the degradation products for 48 h with (A) sodium alginate, (B) poly M, (C) poly G. The eluents were detected by measuring the absorbance at 235 nm. The elution volumes of the unsaturated monomer, dimer, trimer, tetramer,

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pentamer and hexamer are 17.35, 16.25, 15.23, 14.38, 13.61 and 13.01 mL.

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Fig. 6 ESI-MS analysis of the hydrolysis products for 48 h with (A) sodium alginate, (B) polyM; (C) polyG as substrate. The oligo-uronates are commonly described by their degree of polymerization (DPx). Oligo-uronates with the unsaturated terminal

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uronate are denoted in this study as ΔDPx, and such a trimer would be ΔDP3.

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Fig. 7 ESI-MS analysis of products with (A) tetrasaccharide, (B) pentasaccharide, (C)

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hexasaccharide, (D) heptasaccharide, and (E) octasaccharide as substrate.

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Fig.8 (A) Overall structure of AlyPL6; (B) The structural comparison of AlyPL6 (green) and AlyGC (yellow)，module A and module B represented the N-terminal domain of AlyGC and C-terminal domain of AlyGC, respectively. (C) The sequence

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alignments of AlyPL6 and AlyGC.

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Fig. 9 (A) The thermal stability of immobilized AlyPL6 and free AlyPL6; (B) The

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reusability of immobilized AlyPL6.

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Journal Pre-proof Highlights

1. A novel alginate lyase of PL6 family with high activity was identified. 2. The degradation pattern and distribution products of AlyPL6 were analyzed. 3. The enzyme was immobilized onto the mesoporous titanium oxide particles.

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4. The thermal stability and reusability of immobilized enzyme were characterized.

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