Saccharification of alginate by using exolytic oligoalginate lyase from marine bacterium Sphingomonas sp. MJ-3

Journal of Industrial and Engineering Chemistry 17 (2011) 853–858

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Saccharification of alginate by using exolytic oligoalginate lyase from marine bacterium Sphingomonas sp. MJ-3 Mili Ryu, Eun Yeol Lee * Department of Chemical Engineering, Kyung Hee University, Gyeonggi-do 446-701, Republic of Korea

A R T I C L E I N F O

A B S T R A C T

Article history: Received 12 July 2011 Accepted 5 August 2011 Available online 1 October 2011

Alginate is a linear polysaccharide that is abundant in algal biomass. A novel recombinant exolytic oligoalginate lyase from a marine bacterium, Sphingomonas sp. MJ-3, was used for the saccharification of alginate into alginate monosaccharides in order to use alginate monosaccharides as renewable carbon source. The optimal heterologous expression condition for the MJ-3 oligoalginate lyase was determined, and the effects of saccharification reaction conditions were evaluated. Unsaturated monosaccharides up to 3.3 mg/ml were successfully prepared from 1% (w/v) alginate by using the recombinant oligoalginate lyase of Sphingomonas sp. MJ-3. ß 2011 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.

Keywords: Saccharification Oligoalginate lyase Alginate Unsaturated monosaccharide Sphingomonas sp. MJ-3

1. Introduction Alginate is a linear polysaccharide consisting of a-L-guluronate (G) and its C5 epimer, b-D-mannuronate (M) [1]. Alginate lyase catalyzes a b-elimination reaction and produces unsaturated oligo-uronic acids having 4-deoxy-L-erythro-hex-4-enopyranosyluronic acid (a-keto acid) at the non-reducing end (Fig. 1) [2–5]. Alginate lyase has many applications in therapeutics and biotechnology. It can be used for the production of alginate oligosaccharides from alginate. The resulting alginate oligosaccharides have many biologically active properties useful for therapeutic and biotechnology applications [6–9]. Alginate oligosaccharides have been expected to regulate physiological activity in some plant, animals, bacteria and human [10–12]. Alginate lyase can enhance the efficiency of antibiotic killing of pathogen in biofilms via the degradation of acetylated alginate mucoid [13,14]. Alginate lyase can be classified into two major groups based on their mode of degradation. Endo-type alginate lyase catalyzed the cleavage of glycosidic bonds in alginate through a b-elimination reaction and released unsaturated di- and trisaccharides as main products (Fig. 1) [15]. Exo-type alginate lyase degrades oligosaccharides including di-, tri- and tetrasaccharides, and depolymerizes alginate polymer in exolytic manner [16–19].

* Corresponding author. Tel.: +82 31 201 3839; fax: +82 31 204 8114. E-mail address: [email protected] (E.Y. Lee).

‘ecently, algae have attracted much attention as biomass for the production of biofuels such as biodiesel and bioethanol [20,21]. For the production of bioethanol, carbohydrate is extracted from macroalgae such as brown seaweed and then used for the carbon substrate for bioethanol fermentation. Around 30–50% of brown seaweed is carbohydrate, and alginate constitutes 14–37% of seaweed biomass, indicating that alginate is abundant. Up to date, only laminaran and mannitol are used for bioethanol fermentation, after pretreatment for those carbohydrates. When alginate is used as the renewable source for the production of biofuels and chemicals, the saccharification of alginate is prerequisite and alginate lyases can play important roles in the saccharification process [22]. For an efficient saccharification of alginate, a synergistic degradation by the endo- and exo-type alginate lyase is required. Recently, we have cloned and characterized an oligoalginate lyase responsible for the complete degradation of alginate oligosaccharides [23]. The MJ-3 oligoalginate lyase was cloned from a marine bacterium, Sphingomonas sp. MJ-3. Although alginate is one of the abundant carbohydrates, there have been no systematic investigations on enzymatic saccharification of alginate. In this study, we investigated saccharification of alginate by using the recombinant MJ-3 oligoalginate lyase. The heterologous expression of MJ-3 oligoalginate lyase gene in Escherichia coli was optimized. The effects of various reaction conditions such as temperature, pH and enzyme to substrate ratio on saccharification were analyzed and optimized. Batch saccharification of alginate was carried out by using the recombinant MJ-3 oligoalginate lyase.

1226-086X/$ – see front matter ß 2011 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jiec.2011.08.001

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Fig. 1. Alginate lyase-catalyzed degradation of alginate. (A) Endolytic alginate lyase degrades alginate to oligosaccharides in endolytic fashion. (B) Exolytic oligoalginate lyase degrades alginate to monosaccharide in exolytic manner. Both of endo- and exolytic alginate lyase degrade alginate via b-elimination reaction. The unsaturated monomer can be converted non-enzymatically to 4-deoxy-L-erythro-5-hexoseulose uronic acid.

2. Materials and methods 2.1. Cloning and functional expression of MJ-3 exolytic oligoalginate lyase gene The recombinant plasmid containing MJ-3 oligoalginate lyase gene (pColdI/MJ-3) was expressed in E. coli BL21 (DE3). The recombinant E. coli was cultured on a LB medium supplemented with 50 mg/ml ampicillin and 34 mg chloramphenicol/ml for 8 h at 180 rpm and 37 8C. After cell culture for 8 h, 1 mM IPTG was added for expressing the MJ-3 oligoalginate lyase gene, and incubated at 15 8C for 12 h to express the target protein. 2.2. SDS-PAGE and immunoblotting analysis of MJ-3 exolytic oligoalginate lyase protein The recombinant E. coli cells expressing MJ-3 oligoalginate lyase gene at low temperature were harvested by centrifugation, and resuspended in 50 mM sodium phosphate lysis buffer containing 300 mM NaCl, 10 mM imidazole, 10% (v/v) glycerol and 0.5% (v/v) Triton X-100. Lysozyme and phenylmethanesulfonyl fluoride were added to the final concentrations of 1 mg/ml and 1 mM, respectively. The suspension solution was homogenized by ultrasonicator for 20 min with ice cooling. Then, the recombinant lysate was incubated at 37 8C for 10 min. The cell debris was precipitated by centrifugation (13,000 rpm, 20 min) at 4 8C, and the supernatant was filtered by a membrane with a cut-off 0.2 mm to obtain a clear extract. The precipitate was re-suspended in the lysis buffer to get insoluble proteins after washing the cell debris with the same buffer. To analyze the effect of culture conditions on the expression of soluble proteins, the lysate, soluble and insoluble protein fractions were prepared at 95 8C for 10 min with Laemmli

buffer. The proteins were separated on 12% (v/v) SDS-polyacrylamide gel, and then transferred onto a nitrocellulose membrane. The membrane was incubated with polyclonal antibody against hexahistidine (H-15, Santa Cruz Biotechnology Inc.) and peroxidase-conjugated anti-rabbit IgG (Jackson Immunoresearch), and then visualized with CN/DAB (4-chloronaphthol/3,30 -diaminobenxidine) Substrate Kit (Pierce, USA). 2.3. Alginate-degrading activity assay for MJ-3 oligoalginate lyase Batch degradation reaction was carried out in 20 mM phosphate buffer containing various amounts of sodium alginate at given reaction temperature and pH. The formation of alginate degradation products was analyzed by measuring the absorbance at 235 nm or by thiobarbituric acid (TBA) assay at 548 nm [15]. For the quantitative analysis of a-keto acid (4-deoxy-L-erythro-5hexoseulose uronic acid), 2-deoxy-D-glucose was used as a standard, because deoxy sugars reacted with TBA. The reaction products were analyzed by TLC with a solvent of 1butanol:formic acid:water (4:6:1, v/v/v) and compared with the authentic mixture of dimer and trimer prepared by BioGel P2 gelfiltration chromatography (2.5 cm  240 cm, BioRad, USA) [15]. The depolymerized products were visualized by spraying 10% (v/v) sulfuric acid in ethanol. For visualization of a-keto acids in TLC, TBA was used [18]. 2.4. Effects of pH, temperature and divalent metal ions on alginatedegrading activity of MJ-3 oligoalginate lyase Batch degradation reactions at different pH and temperature were conducted to analyze the effects of pH and temperature on alginate-degrading activity of MJ-3 oligoalginate lyase. The

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Fig. 2. SDS-PAGE and immunoblotting analysis of the recombinant MJ-3 oligoalginate lyase in E. coil BL21 (DE3). Left-hand side: SDS-PAGE, right-hand side: immunoblotting. M indicates standard marker. Lane 1, recombinant cell without IPTG induction at 15 8C; lane 2, recombinant cell with IPTG induction at 15 8C; lane 3, recombinant cell lysate without IPTG induction at 15 8C; lane 4, recombinant cell lysate with IPTG induction at 15 8C; lane 5, recombinant cell without IPTG induction at 37 8C; lane 6, recombinant cell with IPTG induction at 37 8C; lane 7, recombinant cell lysate without IPTG induction at 37 8C; lane 8, recombinant cell lysate with IPTG induction at 37 8C. A 30 mg of cell or lysate was used.

temperature range was from 30 to 60 8C. Four types of 10 mM buffers, acetate buffer (pH 3.6–5.8), potassium phosphate buffer (pH 5.6–8.0), Tris–HCl buffer (pH 7.0–10.0) and sodium carbonate buffer (pH 9.0–11.0), were used to adjust the pH of the reaction solution. In order to evaluate the effects of metal ion for MJ-3 oligoalginate lyase activity, the purified lyase enzyme was incubated with final concentration of 2 mM salt solution including NaCl, ZnCl2, CaCl2, MgCl2, HgCl2, KCl, CoCl2, MnCl2, BaCl2, CuCl2, and FeCl2 in 20 mM phosphate buffer for 10 min at 37 8C before starting the batch reaction for 10 min. Aliquots of the reaction solution were periodically withdrawn to follow the time-course of the saccharification of alginate by MJ-3 oligoalginate lyase. 2.5. Batch saccharification of alginate by using Sphingomonas sp. MJ3 oligoalginate lyase Batch saccharification of alginate by Sphingomonas sp. MJ-3 oligoalginate lyase or recombinant lysate was carried out in 1 ml 20 mM KH2PO4 buffer in 2 ml screw-cap bottles. 1% (w/v) alginate was degraded in the presence of 200 mg/ml lysate at 40 8C and 180 rpm. The progressions of batch saccharification were followed by the analysis of samples withdrawn periodically from the reaction mixture by using TBA analysis. 2.6. Chemicals All chemicals alginate were used as purchased without any purification. Sodium alginate (3500 cps grade, extracted from brown algae) was purchased from Sigma Co. (USA). 3. Results and discussion 3.1. Optimal expression of recombinant oligoalginate lyase of Sphingomonas sp. MJ-3 The MJ-3 oligoalginate lyase has a molecular mass of 79.9 kDa. The size of MJ-3 oligoalginate lyase is rather larger than other types of alginate lyases. Hence, the expression of MJ-3 oligoalginate lyase

in E. coli strain may be not a straightforward job. In order to express the MJ-3 oligoalginate lyase efficiently, pColdI expression vector was employed because the expression of the target gene can be functionally induced and expressed at low temperature. In general, protein expression at low temperature provides the advantage that slow translation at low temperature is generally good for functional expression and appropriate folding of foreign protein in E. coli. The recombinant pColdI/MJ-3 plasmid was transformed to E. coli strains BL21 (DE3), and then expressed at 37 and 15 8C, respectively, in order to investigate the effect of expression temperature on the expression of MJ-3 oligoalginate lyase. When same amounts of cell lysate were analyzed by SDS-PAGE, the expression levels of the target protein were similar for both of the recombinant cells expressing the target gene at 37 and 15 8C (Fig. 2). However, in terms of alginate-degrading activity on the basis of same amount of cell lysate, the specific activity of cell lysate at 15 8C was 1.5-fold higher than that at 37 8C, although the expression levels of the gene were similar each other. This result indicates that the relative ratio of MJ-3 oligoalginate lyase in active form to total recombinant proteins might be high at 15 8C, compared to 37 8C. Based on the above result, we employed the recombinant MJ-3 oligoalginate lyase expressed at low temperature as the biocatalyst for the saccharification of alginate. 3.2. Identification of degradation product by the recombinant MJ-3 oligoalginate lyase The batch degradation pattern was monitored by using FPLC and TLC analysis. The degradation product mixture of alginate by the recombinant MJ-3 oligoalginate lyase at 1 and 30 min were analyzed by FPLC (Fig. 3(A)). As the saccharification reaction progressed, the oligomers such as dimer and trimer gradually disappeared. An authentic mixture sample of di and trisaccharides was generated by endo-type alginate lyase from Streptomyces sp. ALG-5 [15], and loaded on TLC plate as the reference for molecular mass comparison. Although the end product, alginate monosaccharide, could not be clearly visualized by TLC, the spots for di- and trisaccharides gradually disappeared (data not shown). As shown

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Fig. 3. FPLC (A) chromatogram and ESI-MS (B) analysis of the degradation products of alginate by the recombinant MJ-3 oligoalginate lyase. A 1% (w/v) alginate in 20 mM phosphate buffer (pH 7.2) with 20 mg MJ-3 oligoalginate lyase as the biocatalyst was incubated at 30 8C. (A) FPLC chromatogram of the reaction products at 1 and 30 min. The elution peaks of unsaturated monomer, dimer, trimer, tetramer and polymer are indicated in the figure. (B) The peak at 138 was the noisy peak from buffer. The molecular peak of 175 Da was detected as the corresponding alginate monosaccharide.

in Fig. 3(A), the alginate substrate was almost completely depolymerized into monosaccharides by the MJ-3 oligoalginate lyase, clearly indicating that the recombinant MJ-3 oligoalginate lyase could completely degrade alginate into monosaccharide. The molecular mass ([MH]) of the resulting monosaccharide, unsaturated uronic acid, was determined to be 175 Da by ESI-MS analysis (Figs. 1 and 3(B)). Since the recombinant MJ-3 oligoalginate lyase possesses b-elimination activity, unsaturated monosaccharide with molecular mass of 176 Da was formed finally (Fig. 1). 3.3. Effect of metal ions, pH and temperature on the activity of recombinant oligoalginate lyase of Sphingomonas sp. MJ-3 In order to optimize the saccharification reaction of alginate polymer, we conducted optimization experiment. Previously, when we prepared alginate oligomer by using endo-type alginate lyase from Streptomyces sp. ALG-5, there was increase in activity of ALG-5 alginate lyase in the presence of divalent metal ions [15]. We investigated the effect of addition of metal ions on alginate saccharification activity of the recombinant MJ-3 oligoalginate lyase. As shown in Fig. 4, the presence of NaCl, CaCl2, KCl and BaCl2 increased the alginate-degrading activity of MJ-3 oligoalginate lyase. When 1 mM CaCl2 or BaCl2 was added the reaction solution, 1.2-fold increase in the activity of the recombinant MJ-3 oligoalginate lyase, representing that the presence of divalent metal ions increased the alginate-degrading activity. Similar

pattern of activity enhancement in the presence of Ca2+, Mn2+, Fe2+ and Mg2+ at 1 mM was observed for the oligoalginate lyase of Sphingomonas sp. strain A1 [16]. However, the presence of 1 mM Cu2+ and Zn2+ strongly inhibited the MJ-3 oligoalginate lyase activity. The reason why these divalent cations inhibited the activity of the recombinant MJ-3 oligoalginate lyase remains to be elucidated. The effects of reaction temperature and pH on alginatedegrading activity of the recombinant MJ-3 oligoalginate lyase were analyzed and optimized. The pH of the reaction buffer was changed from pH 6.0 to 7.5, and the batch saccharification reactions were carried out. The pH 6.5 was the best for the activity of recombinant MJ-3 oligoalginate lyase (Fig. 5). The reaction temperature was varied in the range from 20 to 80 8C, and the recombinant MJ-3 oligoalginate lyase exhibited maximum alginate-degrading activity at 30 8C (Fig. 6(A)). The saccharification conversion at 30 8C was higher than that of 40 8C at initial stage of saccharification, but both saccharification curves showed saturation after 800 min. When the enzyme stability was evaluated at different temperature, the stability was well maintained up to 40 8C. The enzyme stability was not good at the temperature above 50 8C (Fig. 6(B)). Based on the above results, the optimal temperature was determined to be 30 8C because the recombinant MJ-3 oligoalginate lyase showed a maximum activity at 30 8C and the recombinant enzyme stability was relatively well maintained at 30 8C.

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3.4. Effect of biocatalyst to substrate ratio on the saccharification of alginate Effects of enzyme to substrate ratio on the saccharification of alginate were investigated (Fig. 7). The amount of purified recombinant alginate lyase varied from 2 to 30 mg/ml. The degradation rates at the beginning of the reaction increased with increasing enzyme concentration, whereas the maximum obtainable amount of degradation products was not significantly different except the case of 2 mg/ml enzyme. The time-courses of the batch saccharification against enzyme concentrations were obtained as saturation curves. For quantitative analysis of production of unsaturated monosaccharides, we represented the concentration of unsaturated monosaccharides as 2-deoxy-D-glucose equivalent. About 2.6 mg/ml of monosaccharides with 26% saccharification conversion was obtained from 1% (w/v) alginate by the purified recombinant alginate lyase more than 5 mg/ml. After the characterization of the purified enzyme, we employed the recombinant cell lysate containing the recombinant MJ-3 oligoalginate lyase because purification of enzyme causes additional cost for the saccharification process. Effects of lysate to substrate ratio were analyzed to determine optimal amount of [(Fig._5)TD$IG]

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Fig. 4. Metal ion effect on the alginate-degrading activity of Sphingomonas sp. MJ-3 oligoalginate lyase. A 1% (w/v) alginate in 20 mM phosphate buffer (pH 7.2) with 20 mg MJ-3 oligoalginate lyase as the biocatalyst was incubated at 30 8C in the presence of various metal ions. The concentration of the metal ions was 1 mM. The relative activity was calculated on the basis of initial saccharification rate in the absence of any metal ion as 100%.

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Temperature (oC) Fig. 6. Effect of temperature on the saccharification of alginate by Sphingomonas sp. MJ-3 oligoalginate lyase (A) and stability of MJ-3 oligoalginate lyase (B). A 1% (w/v) alginate in 20 mM phosphate buffer (pH 6.5) with 20 mg MJ-3 oligoalginate lyase as the biocatalyst was incubated at different temperatures. (Symbols: -*-, 30 8C; -*-, 40 8C; -!-, 50 8C; -D-, 60 8C.)

lysate (Fig. 8). We used 1% (w/v) of sodium alginate, and then various amounts of the recombinant cell lysate containing MJ-3 oligoalginate lyase were added to the reaction solution. As the amount of lysate increased, the initial rate of the saccharification [(Fig._7)TD$IG] increased, as expected (Fig. 8(A)). When more than 0.2 mg/ml of 3.0

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Time (min) Fig. 5. pH effect on the saccharification of alginate by Sphingomonas sp. MJ-3 oligoalginate lyase. A 1% (w/v) alginate in 20 mM phosphate buffer (pH 7.2) with 20 mg MJ-3 oligoalginate lyase as the biocatalyst was incubated (30 8C) at different pH. (Symbols: -*-, pH 6.0; -*-, pH 6.5; -!-, pH 7.0; -D-, pH 7.5.)

Fig. 7. Effect of the MJ-3 oligoalginate lyase concentration on saccharification of alginate. A 1% (w/v) alginate in 20 mM phosphate buffer (pH 6.5) with different amounts of MJ-3 oligoalginate lyase as the biocatalyst was incubated at 30 8C. (Symbols: -*-, 2 mg/ml; -*-, 5 mg/ml; -!-, 10 mg/ml; -D-, 20 mg/ml; -&-, 30 mg/ ml.)

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lysate was used, similar patterns of the alginate saccharification were obtained and conversions were also in similar level (Fig. 8(B)). Hence, 0.2 mg/ml of lysate was set as the optimal concentration of lysate for a batch saccharification of alginate. 3.5. Batch saccharification of alginate by using Sphingomonas sp. MJ3 oligoalginate lyase Batch saccharification of 1% (w/v) alginate was conducted at the optimized condition, pH 6.5 and 30 8C. A 0.2 mg/ml of the recombinant cell lysate containing MJ-3 oligoalginate lyase was [(Fig._8)TD$IG]used as the biocatalyst. As the saccharification reaction proceeded,

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the apparent viscosity of the reaction solution decreased, representing that saccharification of alginate occurred. The saccharification products did not include alginate oligosaccharides such as di or trisaccharides because the recombinant MJ-3 alginate lyase degraded alginate exolytically like A1 or Atu3025 alginate lyases [16,18,23]. As shown in Fig. 8(C), the amount of the monosaccharides presented as 2-deoxy-D-glucose equivalent increased as alginate substrate was degraded by the recombinant alginate lyase. The unsaturated monosaccharide concentration increased up to 2.3 mg/ ml of equivalent 2-deoxy-D-glucose from 1% (w/v) alginate. In terms of conversion, around 3 g/l of saccharification products was obtained from 1% (w/v) of initial alginate through alginate lyase-catalyzed saccharification. In order to enhance the conversion, we added additional recombinant cell lysate containing MJ-3 oligoalginate lyase during the saccharification, but there was only small increase in the conversion (data not shown). There might be product inhibition during the saccharification. Additional experiments on the mechanistic analysis on the reason why there is no increased monomer produced above 3 g/l are under investigation.

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Batch saccharification of alginate was investigated by using recombinant oligoalginate lyase from Sphingomonas sp. MJ-3 that possesses an exolytic oligoalginate lyase activity. The heterologous expression conditions and effects of saccharification reaction conditions were analyzed and optimized. Mixtures of unsaturated monosaccharides corresponding to 2.3–3.3 mg/ml of equivalent 2deoxy glucose were obtained at 30 8C and pH 6.5 by using 200 mg/ ml of recombinant lysate or 20 mg/ml of the purified recombinant oligoalginate lyase. The development of alginate lyase and relevant bioprocess for alginate saccharification will be crucial for the application of alginate as renewable biomass for preparing bioactive materials and other biochemicals. Acknowledgement

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This work was supported by a grant from the Kyung Hee University in 2010 (KHU-20100629).

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Time (min) Fig. 8. Effect of recombinant lysate concentration on initial degradation rate (A) and relative conversion on the saccharification of alginate (B), and time-course of batch saccharification (C) by using the recombinant lysate containing the recombinant MJ-3 oligoalginate lyase. A 1% (w/v) alginate in 20 mM phosphate buffer (pH 6.5) with different amounts of recombinant lysate as the biocatalyst was incubated at 30 8C.

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