TY - JOUR
AU - Ohshiro, Takashi
AB - Abstract Alginate, which is an anionic polysaccharide, is widely distributed in the cell wall of brown algae. Alginate and the products of its degradation (oligosaccharides) are used in stabilizers, thickeners and gelling agents, especially in the food industry. The degradation of alginate generally involves a combination of several alginate lyases (exo-type, endo-type and oligoalginate lyase). Enhancing the efficiency of the production of alginate degradation products may require the identification of novel alginate lyases with unique characteristics. In this study, we isolated an alginate-utilizing bacterium, Shewanella sp. YH1, from seawater collected off the coast of Tottori prefecture, Japan. The detected novel alginate lyase was named AlgSI-PL7, and was classified in polysaccharide lyase family 7. The enzyme was purified from Shewanella sp. YH1 and a recombinant AlgSI-PL7 was produced in Escherichia coli. The optimal temperature and pH for enzyme activity were around 45°C and 8, respectively. Interestingly, we observed that AlgSI-PL7 was not thermotolerant, but could refold to its active form following an almost complete denaturation at approximately 60°C. Moreover, the degradation of alginate by AlgSI-PL7 produced two to five oligosaccharides, implying this enzyme was an endo-type lyase. Our findings suggest that AlgSI-PL7 may be useful as an industrial enzyme. alginate lyase, characterization, conformation, PL family 7, purification Polysaccharides derived from seaweeds are useful in various fields (1–3). Specifically, polysaccharides and oligosaccharides are widely used in the commercial food and pharmaceutical industries (4–6). Additionally, there is growing interest in using algal biomass as an alternative to fossil fuels as an energy resource (7–9). Alginate, which is a linear acidic polysaccharide, is a major cell wall component in brown macroalgae, representing about 30% of the dry weight of these seaweeds (4). Alginate consists of β-D-mannuronic acid (M) linked to its C-5 epimer α-L-guluronic acid (G) with 1,4-o-glycoside bonds. Moreover, alginate consists of two homopolymeric regions [i.e. polyguluronic acid (polyG) and polymannuronic acid (polyM) regions] and one heteropolymeric region [i.e. random region (polyMG)] (4, 10). The polyG region binds divalent cations and can form strong but reversible alginate gels, while the polyM and polyMG regions form weak elastic alginate gels (10, 11). Furthermore, two bacterial genera, Pseudomonas and Azotobacter, can synthesize alginate during their vegetative growth phase (4, 12–14). Alginate has been used as a stabilizer, viscosifier and gelling agent, as well as in films and therapeutic agents (4, 6). Moreover, the degradation of alginate generates oligosaccharides with physiological functions and biological activities, including plant growth-promoting activities as well as anti-oxidant, anti-coagulant, anti-tumor, anti-hypersensitive, anti-proliferative, and anti-allergy properties (15–23). However, enzymatic digestions are required to obtain bioactive oligosaccharides. The low molecular weight alginate produced by acid hydrolysis is significantly less active than the products resulting from the enzymatic digestion of an alginate oligomer (16, 24). Consequently, characterizing the alginate-degrading enzymes may have important implications for the food and pharmaceutical industries. Polysaccharide lyases (PLs) have been classified into 27 families in the Carbohydrate-Active enZYmes Database (http://www.cazy.org). Alginate lyases belong to PL families 5–7, 14, 15, 17, and 18 (25). These enzymes catalyze the depolymerization of alginate through a β-elimination of the 4-O-glycosidic bonds, which is accompanied by the formation of a double bond between C-4 and C-5 (26). Alginate lyases are generally divided into two groups based on substrate specificity. The polyG lyases (EC 4.2.2.11) specifically target the polyG and polyMG regions, while the polyM lyases (EC 4.2.2.3) specifically target the polyM region. These enzymes function in an exolytic or endolytic manner. Although an EC number has not been assigned, some alginate lyases were recently observed to be active against both polyG and polyM regions (i.e. bifunctional). This implies that these enzymes degrade alginate more effectively than other enzymes with similar functions to produce alginate oligomers (27–29). To date, more than 50 alginate lyases have been isolated from various sources (e.g. marine algae, marine mollusks, fungi, bacteria, bacteriophages, and viruses) and subsequently characterized (26, 30). The degradation of alginate by marine bacteria is mediated by various alginate lyases involved in extra- and inter-cellular degradation pathways. First, alginate lyases belonging to PL families 5 and 7 degrade alginate endolytically to produce oligosaccharides. Second, oligoalginate lyases from PL families 6, 15 and 17 exolytically degrade the oligosaccharides to form monosaccharides (31). Finally, the generated monosaccharides are converted to 4-deoxy-l-erythro-5-hexoseulose uronic acid via a metabolic system. Interestingly, recent studies revealed that alginate lyases have a broad substrate specificity and can tolerate heat, salt and cold stresses (32–34). Although these characteristics are important for producing oligosaccharides, enzyme stability and refolding ability are also key considerations, particularly for industrial applications. Refolding ability is especially important in terms of purifying or recycling of enzymes. In this study, we focused on marine bacterial alginate lyases that are able to refold. We isolated a novel alginate lyase (AlgSI-PL7) from the marine bacterium, Shewanella sp. YH1. Interestingly, although AlgSI-PL7 aggregated and lacked lyase activities after a 1-h treatment at 40°C, its activities were recovered following an incubation at 60°C. Additionally, AlgSI-PL7 was observed to endolytically degrade alginate into two to five oligosaccharides. Our findings suggest that AlgSI-PL7 may be useful as an industrial enzyme for the production of oligosaccharides. Materials and Methods Screening of marine bacteria that produce alginate-degrading enzymes To search for marine bacteria that produce alginate-degrading enzymes, we analysed seawater collected from the seacoast in Tottori prefecture, Japan. A 200-µl sample of seawater was added to culture medium [23 mm K2HPO4, 3.7 mm KH2PO4, 1.7 mm MgSO4, 15 mm (NH4)2SO4, 0.5 m NaCl (3%, w/v), metal solution and vitamin mixture] supplemented with 0.5% (w/v) sodium alginate (1000 cps) (Nacalai Tesque Inc., Japan) as the sole carbon source (alginate medium). The marine bacteria were cultured at 30°C. Pure strains obtained by repeating an enrichment culture and agar plate dilution method were identified based on a 16S rRNA phylogenetic analysis. Purification of an enzyme from a marine bacterium Shewanella sp. YH1 cells in alginate medium were cultured at 20°C for 36 h with shaking (180 rpm). Cells were harvested and sonicated in 50 mm Tris–HCl buffer (pH 7.0). After centrifuging the lysed cells, streptomycin sulfate (2.5% final) was added to the supernatant to remove nucleic acids. The sample was centrifuged and the supernatant was dialyzed in 50 mM Tris–HCl (pH 7.0) before being applied to a Resource Q column (GE Healthcare, UK). Samples were eluted with a linear gradient of 0–1.0 m NaCl. The collected protein fractions were applied to a Superdex 75 column (GE Healthcare). After a dialysis, the samples were re-applied to a Resource Q column. The concentration of the purified AlgSI-PL7 was determined using the Bradford method (Bio-Rad Laboratories, USA). N-terminal amino acid sequence The purified AlgSI-PL7 was analysed by a 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride membrane. The N-terminal amino acid sequence was determined using the PPSQ-30 protein sequencer (Shimadzu Corp., Japan). Enzymatic activity assay A 1-ml reaction solution containing 0.1 m Tris–HCl buffer (pH 7.0), 0.2% (v/v) substrate [sodium alginate (1000 cps), polyMG, polyM and polyG] and 5–10 µl (0.02–0.04 U) enzyme was prepared to analyse lyase activity. PolyMG, polyM and polyG were prepared as previously described (10). Lyase activity was measured at 42°C for 3 min, and the absorbance at 232 nm was recorded. Following the enzymatic degradation of alginate to oligosaccharides, the products of the non-reducing end of uronic acids have a double bond between C4 and C5. Because the double bond absorbs strongly at 232 nm, this wavelength can be used to evaluate enzyme activity. Experiments were completed to optimize the temperature (20–60°C), pH (5.5–11), effects of metal ions (1 mM) and inhibitors (1 mm) and substrate specificity (polyG, polyM and polyMG). For the thermostability experiment, samples were pre-incubated at 10–90°C for 1 h and then cooled on ice for at least 15 min. Samples were analysed at 42°C using a UV-2600 spectrophotometer (Shimadzu Corp.). The absorbance at 232 nm increased by 5.5 when 1 mm unsaturated uronic acids was produced. One unit of lyase activity was defined as the amount of enzyme required to produce 1 µmol oligosaccharides containing unsaturated uronic acids per minute. Recombinant AlgSI-PL7 (rAlgSI-PL7) was produced in inclusion bodies (i.e. insoluble fraction) in an E. coli BL21 (DE3) expression system. A cell-free extract containing rAlgSI-PL7 and 8 m urea was dialyzed to refold the enzyme. Mass spectrometry The molecular weight of the purified enzyme was determined with an AutoFLEX MALDI-TOF mass spectrometer (Bruker, USA). Samples were prepared using C-18 Zip-tips and mixed with sinapic acid (Sigma-Aldrich, USA), which served as the MALDI matrix. The mass spectrometer was operated in the linear mode. The products resulting from the degradation of sodium alginate were examined by electrospray ionization mass spectrometry, with the resulting data analysed by the Xcalibur program (Thermo Fisher Scientific, USA). The mass spectrometer was operated in the negative mode. To prepare samples, reaction mixtures were incubated at 30°C for 48 h. After a centrifugation, the samples were applied to a Superdex peptide column (GE Healthcare). The degradation products, AlgSI-PL7, and undigested sodium alginate were separated based on the absorbance at 232 nm. The collected fractions were applied to an Amide-80 column (Tosoh, Japan) and samples were eluted with a linear gradient of 95–70% acetonitrile. Cloning and expression of recombinant AlgSI-PL7 The algsi-pl7 gene was identified during the analysis of the Shewanella sp. YH1 genome using the HiSeq 2500 high-throughput sequencing system (Illumina Inc., USA). We designed polymerase chain reaction primers to amplify algsi-pl7 (forward primer 5′-GGAGATATACATATGTCAATTGAACTTGATACAGCC-3′; reverse primer 5′-GTGGTGGTGCTCGAGTTACTGCTTTAACTCAAGTTG-3′). Additionally, algsi-pl7 was ligated into the pET-21a expression vector according to the In-Fusion HD cloning method (Takara Bio Inc., Japan) for a subsequent transformation of E. coli HST08 premium competent cells (Takara Bio Inc.). To produce rAlgSI-PL7, the purified pET-21a–algsi-pl7 vector was inserted into E. coli BL21 (DE3) and SHuffle cells, which were then cultured at 37 and 20°C, respectively, in LB broth containing ampicillin. We added 1 mm (final concentration) isopropyl β-d-1-thiogalactopyranoside to the bacterial culture to induce the expression of rAlgSI-PL7, which lacked a signal sequence (33). The rAlgSI-PL7 was produced in inclusion bodies in BL21 (DE3) cells, whereas 50% of the rAlgSI-PL7 were in the soluble fraction of SHuffle cells. Refolding assay Along with rAlgSI-PL7, rAlgC-PL7, which is an alginate lyase derived from Cobetia sp. NAP1, was produced in an E. coli BL21 (DE3) expression system (33). Both rAlgSI-PL7 and rAlgC-PL7 were produced as insoluble enzymes, which were denatured (i.e. unfolded) and resolubilized in 50 mm Tris–HCl (pH 7.0), 0.1 mm DTT, 0.1 mm EDTA and 8 m urea. We added 5-µl samples of the unfolded enzymes to 995 µl lyase assay buffer, resulting in a 200-fold dilution of the urea concentration. Thus, rAlgSI-PL7 and rAlgC-PL7 were able to refold. Circular dichroism (CD) analysis The far-UV CD spectrum of AlgSI-PL7 was obtained at various temperatures (25, 40, and 70°C) with the J-820 spectropolarimeter (Jasco, Japan) using a cell with a light path of 1 mm under the desired solvent conditions. Samples were prepared in 5 mm Tris–HCl buffer (pH 7.0) and were analysed at 0.38 mg/ml. The CD signals between 195 and 250 nm were expressed as the mean residue ellipticity [θ] (deg cm2 dmol−1). The temperature was regulated using a PTC-423L Peltier unit (Jasco). Aggregation assay A thioflavin T (ThT) fluorescence assay was conducted to assess the aggregation of AlgSI-PL7. Samples were incubated at 40 and 70°C for 1 and 24 h. A 10-µl aliquot of each sample was added to 1 ml ThT solution (10 µM ThT in 50 mM glycine-NaOH, pH 8.0) and then analysed using the FP-8300 spectrofluorometer (Jasco) with excitation and emission wavelengths of 440 and 450–550 nm, respectively. Results Screening of Shewanella sp. YH1 Alginate-degrading bacteria were isolated from seawater collected off the coast of Tottori prefecture, Japan. Following repeated screenings and colony isolations, we identified the bacteria based on a 16S rRNA phylogenetic analysis completed using extracted genomic material. The alginate-degrading bacteria were all identified as the Gram negative marine bacterium, Shewanella sp., which we named Shewanella sp. YH1. Although we screened alginate-degrading bacteria using cultures grown at 30°C, the optimal temperature for Shewanella sp. YH1 growth was 20°C. Thus, cells were cultured at 20°C during the purification of the alginate-degrading enzyme from the wild-type bacterial strain. Purification and analysis of the alginate-degrading enzyme from Shewanella sp. YH1 We purified and characterized an alginate-degrading enzyme from a wild-type strain of Shewanella sp. YH1. However, prior to the purification, we examined the effects of aeration on enzyme production in culture. Cell-free extracts were prepared for each condition and tested for enzyme activity. Under low aeration conditions, Shewanella sp. YH1 grew but exhibited relatively low enzyme activity. In contrast, high enzyme activity was observed for bacteria grown under high aeration conditions. A comparison of this difference by SDS-PAGE revealed that the well-aerated bacteria produced an extra band (approximately 35 kDa) (Supplementary Fig. S1A). We subsequently grew Shewanella sp. YH1 under high aeration conditions with either glucose or alginate as the sole carbon source to compare the production of alginate-degrading enzymes. Moreover, we measured the enzyme activity of each cell-free extract, and observed a lack of enzyme activity in the glucose-containing medium (Supplementary Fig. S1B). These results suggested that the production of this protein was induced by alginate. Thus, our attempts at purifying alginate-degrading enzymes focused on proteins that were around 35 kDa. During the purification process, we monitored lyase activity and eventually purified an approximately 35-kDa enzyme (Fig. 1A). Fig. 1 View largeDownload slide SDS-PAGE analysis of AlgSI-PL7 and determination of the N-terminal amino acid sequence. (A) SDS-PAGE analysis of AlgSI-PL7. Lane M, molecular weight markers; lanes 1 and 2, purified AlgSI-PL7 (35 kDa). (B) The AlgSI-PL7 signal sequence analysed using the SignalP Server indicated the enzyme is localized in the periplasm. The N-terminal amino acid sequence was determined for purified native AlgSI-PL7 after the signal sequence was removed. The AlgSI-PL7 enzyme includes three conserved amino acid sequences (RSEL, QIH and YYKAGVYNQ), indicating AlgSI-PL7 belongs to the PL-7 family. Fig. 1 View largeDownload slide SDS-PAGE analysis of AlgSI-PL7 and determination of the N-terminal amino acid sequence. (A) SDS-PAGE analysis of AlgSI-PL7. Lane M, molecular weight markers; lanes 1 and 2, purified AlgSI-PL7 (35 kDa). (B) The AlgSI-PL7 signal sequence analysed using the SignalP Server indicated the enzyme is localized in the periplasm. The N-terminal amino acid sequence was determined for purified native AlgSI-PL7 after the signal sequence was removed. The AlgSI-PL7 enzyme includes three conserved amino acid sequences (RSEL, QIH and YYKAGVYNQ), indicating AlgSI-PL7 belongs to the PL-7 family. Edman degradation revealed that the first 12 amino acids at the N-terminal of the purified alginate-degrading enzyme were SIELDTAKQFNL (Fig. 1B). Additionally, we analysed the Shewanella sp. YH1 genome using next-generation sequencing technology. The resulting sequencing data enabled us to determine the complete amino acid sequence of the purified enzyme (Fig. 1B). Because of the detection of an additional N-terminal sequence, we examined the enzyme using the SignalP 4.1 Server (http://www.cbs.dtu.dk/services/SignalP/). Consequently, we determined that the enzyme is produced with a signal peptide and is localized in the periplasm (Fig. 1B). Furthermore, according to a BLAST search using the amino acid sequence as a template (https://blast.ncbi.nlm.nih.gov), the purified alginate-degrading enzyme belongs to PL family 7. Thus, we named the purified enzyme AlgSI-PL7. The theoretical pI and molecular weight of AlgSI-PL7 after the signal peptide has been removed were 6.01 and 36376.43 Da, respectively, which was consistent with the SDS-PAGE and MALDI-TOF mass spectral data. Our findings indicated that we purified an alginate lyase from a wild-type strain of Shewanella sp. YH1. Determining the optimal temperature and pH for AlgSI-PL7 activity We examined the effects of temperature and pH on AlgSI-PL7 activity. A lyase activity assay was used to determine the specific activity. To determine the optimal temperature for AlgSI-PL7 activity, we examined the effects of Tris and phosphate buffers at temperatures between 20 and 60°C. Interestingly, enzymatic activity was about two times higher in the phosphate buffer than in the Tris buffer, with an optimal temperature of around 45°C (Fig. 2A). Moreover, although the optimal pH for AlgSI-PL7 activity in the phosphate buffer was around 8.0, the enzyme retained about 70% of its lyase activity between pH 7 and 9 (Fig. 2B). These results suggested that AlgSI-PL7 functions optimally at neutral pH. Fig. 2 View largeDownload slide Effects of temperature, pH and salts on AlgSI-PL7 activity. Optimal temperature (A) and pH (B) for AlgSI-PL7 activity. Lyase activity was measured for AlgSI-PL7 in sodium acetate buffer (pH 6.2), potassium phosphate buffer (pH 5.9–8.6 and 9.5–11) and Tris buffer (pH 5.8–9.5). (C) Salt-dependent AlgSI-PL7 lyase activity. The salt-induced activities are presented relative to the activity in the absence of salt (gray). (D) Thermostability of AlgSI-PL7. Samples were incubated at each temperature for 1 h and then cooled on ice. The assays of salt-dependent activity and thermostability (C and D) were completed with samples prepared in a phosphate buffer. Fig. 2 View largeDownload slide Effects of temperature, pH and salts on AlgSI-PL7 activity. Optimal temperature (A) and pH (B) for AlgSI-PL7 activity. Lyase activity was measured for AlgSI-PL7 in sodium acetate buffer (pH 6.2), potassium phosphate buffer (pH 5.9–8.6 and 9.5–11) and Tris buffer (pH 5.8–9.5). (C) Salt-dependent AlgSI-PL7 lyase activity. The salt-induced activities are presented relative to the activity in the absence of salt (gray). (D) Thermostability of AlgSI-PL7. Samples were incubated at each temperature for 1 h and then cooled on ice. The assays of salt-dependent activity and thermostability (C and D) were completed with samples prepared in a phosphate buffer. Effects of metal ions on AlgSI-PL7 Because metals can enhance the activities of some enzymes, we examined the effects of various metal ions (1 mm) on AlgSI-PL7 lyase activity (Table I). Lyase activity decreased in response to most metal ions, especially Cu2+. The exceptions were Na+, K+ and Li+. In fact, AlgSI-PL7 activity may have even been slightly enhanced by Na+ and K+. Thus, we examined the effects of various concentrations of NaCl and KCl on AlgSI-PL7 lyase activity (Fig. 2C). For both Na+ and K+, 0.2 m induced the highest enzyme activity, suggesting that seawater salt concentrations are optimal for AlgSI-PL7 activity. Additionally, sodium alginate was used to investigate enzyme activity, implying Na+ is critical for AlgSI-PL7. We also examined the effects of inhibitors on AlgSI-PL7 (Table II), and observed that N-bromosuccinimide and EDTA considerably decreased enzyme activity. Our findings indicated that AlgSI-PL7 functions well in the presence of Na+ and K+. Table I. Effects of metal ions on AlgSI-PL7 lyase activity Metal ions  Concentration (mm)  Relative activity (%)  Metal ions  Concentration (mm)  Relative activity (%)  Control  0  100.00 ± 2.65  K+  1  106.37 ± 0.71  Al3+  1  59.01 ± 3.14  Li+  1  94.49 ± 0.63  Ba2+  1  13.37 ± 0.16  Mg2+  1  74.75 ± 1.51  Ca2+  1  35.89 ± 1.80  Mn2+  1  8.28 ± 0.38  Co2+  1  11.65 ± 0.34  Na+  1  96.29 ± 7.10  Cu2+  1  7.62 ± 1.04  Ni2+  1  20.12 ± 0.46  Fe3+  1  59.73 ± 6.69        Metal ions  Concentration (mm)  Relative activity (%)  Metal ions  Concentration (mm)  Relative activity (%)  Control  0  100.00 ± 2.65  K+  1  106.37 ± 0.71  Al3+  1  59.01 ± 3.14  Li+  1  94.49 ± 0.63  Ba2+  1  13.37 ± 0.16  Mg2+  1  74.75 ± 1.51  Ca2+  1  35.89 ± 1.80  Mn2+  1  8.28 ± 0.38  Co2+  1  11.65 ± 0.34  Na+  1  96.29 ± 7.10  Cu2+  1  7.62 ± 1.04  Ni2+  1  20.12 ± 0.46  Fe3+  1  59.73 ± 6.69        Table II. Effects of inhibitors on AlgSI-PL7 lyase activity Inhibitors  Concentration (mm)  Relative activity (%)  Control  0  100.00 ± 1.28  2, 2-bipyridine  1  90.14 ± 2.09  N-bromosuccinimide  1  0.00 ± 0.00  N-ethylmaleimide  1  91.65 ± 0.99  EDTA  1  8.16 ± 0.07  DEPC  1  90.73 ± 3.25  DTNB  1  91.99 ± 0.78  Inhibitors  Concentration (mm)  Relative activity (%)  Control  0  100.00 ± 1.28  2, 2-bipyridine  1  90.14 ± 2.09  N-bromosuccinimide  1  0.00 ± 0.00  N-ethylmaleimide  1  91.65 ± 0.99  EDTA  1  8.16 ± 0.07  DEPC  1  90.73 ± 3.25  DTNB  1  91.99 ± 0.78  Thermostability of AlgSI-PL7 We also examined the relationship between the optimal temperature for enzyme activity and the thermostability of AlgSI-PL7 (Fig. 2D). Enzyme samples were incubated at various temperatures for 1 h and then cooled on ice for at least 15 min. A 1-h incubation at 40°C decreased AlgSI-PL7 activity and caused the enzyme to aggregate and precipitate in solution. Furthermore, AlgSI-PL7 activity could not be recovered. In contrast, after a 1-h incubation at 60°C, alginate lyase activity was approximately 67% of the activity observed after 1-h incubations at 10–30°C. These results suggested that AlgSI-PL7 is not thermostable, but can refold to its active state after being denatured. Refolding ability of AlgSI-PL7 To examine the refolding ability of AlgSI-PL7, we cloned and expressed rAlgSI-PL7 and rAlgC-PL7. The AlgC-PL7 enzyme, derived from Cobetia sp. NAP1, has been characterized and cloned (33), and was classified in PL family 7. Although the sequences of AlgSI-PL7 and AlgC-PL7 are 45% similar, we analysed their refolding abilities to compare these PL family members under the same conditions. Both recombinant enzymes were produced in the precipitated protein (Ppt) fraction (Fig. 3A). The Ppt fractions were resuspended in 50 mm Tris–HCl containing 8 M urea, which caused the precipitated enzymes to unfold (i.e. chemical denaturation). We used these samples for lyase activity measurements. The addition of the unfolded enzymes to the reaction solutions resulted in a 200-fold decrease in urea concentration. The kinetic measurements of lyase activity at 232 nm are presented in Fig. 3B. There was no change to the baseline absorbance for the Ppt fraction of the pET21a vector control cells (dotted line). In contrast, the absorbance for the rAlgC-PL7 Ppt fraction increased slightly after 60 s, implying that rAlgC-PL7 was refolding (dashed line). Interestingly, the lyase activity of the rAlgSI-PL7 Ppt fraction increased sharply after the unfolded rAlgSI-PL7 was added to the reaction solution, indicating that rAlgSI-PL7 was able to rapidly refold to its functional state (solid line). Fig. 3 View largeDownload slide Refolding ability of recombinant AlgSI-PL7 and AlgC-PL7. (A) SDS-PAGE analysis of recombinant AlgSI-PL7 and AlgC-PL7 produced in Escherichia coli BL21 (DE3) cells. Both enzymes were produced in the precipitated protein fraction. (B) Kinetic measurements of lyase activities of recombinant AlgSI-PL7 (solid line) and AlgC-PL7 (dashed line). The pET21a vector was used as the control (dotted line). Fig. 3 View largeDownload slide Refolding ability of recombinant AlgSI-PL7 and AlgC-PL7. (A) SDS-PAGE analysis of recombinant AlgSI-PL7 and AlgC-PL7 produced in Escherichia coli BL21 (DE3) cells. Both enzymes were produced in the precipitated protein fraction. (B) Kinetic measurements of lyase activities of recombinant AlgSI-PL7 (solid line) and AlgC-PL7 (dashed line). The pET21a vector was used as the control (dotted line). We also examined the abundance of aggregated AlgSI-PL7 as well as enzyme secondary structures at 40 and 70°C (thermal denaturation). Because there were no extreme changes in AlgSI-PL7 activity between 60 and 70°C, we performed the experiments at the higher temperature. We conducted a ThT-binding assay to detect β-aggregates and amyloid fibrils (Fig. 4A). The ThT fluorescence at 480 nm increased in response to the incubation at 40°C, implying AlgSI-PL7 had aggregated (thick black line and dashed line). In contrast, ThT fluorescence increased only slightly after the 1-h incubation at 70°C (thick gray dashed line). The ThT fluorescence after a longer incubation at 70°C (24 h) was the same as that of the control (thick grey line and thin black line), suggesting that AlgSI-PL7 was unfolded and did not form any aggregates at 70°C. Fig. 4 View largeDownload slide Effects of different temperatures on the aggregation and secondary structure of AlgSI-PL7. (A) Detection of AlgSI-PL7 aggregates by a thioflavin T-binding assay. Buffer control (dashed black line), AlgSI-PL7 control at 4°C (thin black line), 1-h incubation at 40°C (thick black dashed line), 24-h incubation at 40°C (thick black line), 1-h incubation at 70°C (thick grey dashed line) and 24-h incubation at 70°C (thick grey dashed line). (B) Temperature-dependent AlgSI-PL7 secondary structures based on circular dichroism measurements. AlgSI-PL7 control at 4°C (thin black line), 1-h incubation at 40°C (thick dotted line), 1-h incubation at 70°C (thick dashed line) and cooled at 4°C after a 1-h incubation at 70°C (thick line). Fig. 4 View largeDownload slide Effects of different temperatures on the aggregation and secondary structure of AlgSI-PL7. (A) Detection of AlgSI-PL7 aggregates by a thioflavin T-binding assay. Buffer control (dashed black line), AlgSI-PL7 control at 4°C (thin black line), 1-h incubation at 40°C (thick black dashed line), 24-h incubation at 40°C (thick black line), 1-h incubation at 70°C (thick grey dashed line) and 24-h incubation at 70°C (thick grey dashed line). (B) Temperature-dependent AlgSI-PL7 secondary structures based on circular dichroism measurements. AlgSI-PL7 control at 4°C (thin black line), 1-h incubation at 40°C (thick dotted line), 1-h incubation at 70°C (thick dashed line) and cooled at 4°C after a 1-h incubation at 70°C (thick line). To verify the differences in AlgSI-PL7 at each temperature, we evaluated AlgSI-PL7 secondary structures based on CD measurements (Fig. 4B). The control spectrum indicated AlgSI-PL7 was a β-rich structure (thin line). During the incubation at 40°C, the spectrum intensity decreased and there was a lack of negative peaks, indicating AlgSI-PL7 was denatured (dotted line). The decrease in the spectrum intensity was also because AlgSI-PL7 formed aggregates and precipitated in the cuvette. In contrast, the spectrum during the incubation at 70°C suggested AlgSI-PL7 exhibited a random coil-like structure, implying the enzyme was unfolded (dashed line). These results confirmed that AlgSI-PL7 forms different structures at 40 and 70°C. Because AlgSI-PL7 formed aggregates at 40°C, the enzyme was unable to refold after cooling and remained inactive (data not shown). However, a re-analysis of the enzyme cooled after being incubated at 70°C (thick line) revealed that AlgSI-PL7 had changed from a random coil to a β-rich structure, indicating the enzyme was able to refold. Thus, the partial unfolding of AlgSI-PL7 due to an incubation at 40°C resulted in the formation of aggregates that were unable to refold and recover their lyase activity. In contrast, if AlgSI-PL7 was almost completely unfolded, it could refold and recover its activity. Substrate specificity of AlgSI-PL7 The QIH and QVH sequences, which are conserved among PL-7 family members, affect substrate specificity. Previous studies concluded that QIH is specific for polyG and polyMG regions, while QVH is specific for polyM (30, 35). To confirm the substrate specificity of AlgSI-PL7, we synthesized and purified polyM, polyG and polyMG blocks (10, 33). We examined the specific activities of AlgSI-PL7 and rAlgSI-PL7 against each synthesized block (Fig. 5). As expected, AlgSI-PL7 exhibited high lyase activity against the polyG block (Fig. 5A). We also obtained similar substrate specificity and specific activity results for purified rAlgSI-PL7 (Fig. 5B), which had been produced as a soluble enzyme in E. coli SHuffle cells. These results indicated that native and recombinant enzymes exhibited similar characteristics. Fig. 5 View largeDownload slide Substrate specificities of AlgSI-PL7 and recombinant AlgSI-PL7. Substrate specificities of AlgSI-PL7 (A) and recombinant AlgSI-PL7 produced in Escherichia coli SHuffle cells (B). PolyMG, polyG and polyM were prepared as described by Haug et al. Fig. 5 View largeDownload slide Substrate specificities of AlgSI-PL7 and recombinant AlgSI-PL7. Substrate specificities of AlgSI-PL7 (A) and recombinant AlgSI-PL7 produced in Escherichia coli SHuffle cells (B). PolyMG, polyG and polyM were prepared as described by Haug et al. Detection of sodium alginate degradation products produced by AlgSI-PL7 Several endo- and exo-types of alginate lyases are involved in the degradation of alginate. To clarify whether AlgSI-PL7 is an endo- or exo-type alginate lyase, we examined the products derived from the degradation of sodium alginate by this enzyme. After a 48-h incubation, degraded fractions collected by HPLC were analysed by electrospray ionization mass spectrometry (Fig. 6). Although two major peaks were detected (175.024 and 351.056), the 175.024 peak corresponded to Z = −2 (Fig. 6A). Thus, the smallest degradation product was a disaccharide. However, we also detected tri-, tetra- and pentasaccharides (Fig. 6B), which is consistent with the findings of previous studies (36–38). Our data indicated the degradation of alginate by AlgSI-PL7 results in two to five oligosaccharides, implying that AlgSI-PL7 is an endo-type alginate lyase. Fig. 6 View largeDownload slide ESI-MS analysis of the products generated from the degradation of alginate by AlgSI-PL7. (A) The 175 (Z = −2) and 351 (Z = −1) peaks corresponded to disaccharides derived from alginate (36). (B) Additional oligosaccharides produced from the degradation of alginate (527.052 = tri-, 703.121 = tetra- and 879.152 = pentasaccharides). The mass spectrometer was operated in the negative mode. Fig. 6 View largeDownload slide ESI-MS analysis of the products generated from the degradation of alginate by AlgSI-PL7. (A) The 175 (Z = −2) and 351 (Z = −1) peaks corresponded to disaccharides derived from alginate (36). (B) Additional oligosaccharides produced from the degradation of alginate (527.052 = tri-, 703.121 = tetra- and 879.152 = pentasaccharides). The mass spectrometer was operated in the negative mode. Discussion Shewanella sp. YH1 alginate lyases Many alginate-utilizing bacteria have been isolated, and the alginate lyases from Vibrio sp., Pseudomonas sp. and Sphingomonas sp. have been well studied (30, 31, 39, 40). Although some Shewanella spp. have been isolated, there are relatively few reports describing their alginate lyases (41–44). The PL-6 and PL-17 oligoalginate lyases from Shewanella sp. were recently described (43, 44). In this study, we observed the unique characteristics of an endo-type alginate lyase from the PL-7 family, which we named AlgSI-PL7. Additionally, we purified this enzyme from a wild-type Shewanella sp. strain. However, we were unable to purify any other alginate-degrading enzymes from the bacterium. Thus, we identified other Shewanella sp. YH1 alginate lyase genes based on an analysis of a draft genome sequence, a BLAST search, and a comparison with conserved sequences. Consequently, we determined that Shewanella sp. YH1 produces two PL-6 enzymes [(56 kDa; 51% sequence identity to Vibrio harveyi accession WP 074050300.1) and (84 kDa; 72% sequence identity to Shewanella frigidimarina accession WP 059744768.1)], three PL-7 enzymes [(40 kDa including QIH sequence; 76% sequence identity to Vibrio owensii accession WP 054824086.1), (35 kDa including QVH sequence; 58% sequence identity to Shewanella sp. UCD-KL21 accession WP 076541881.1) and (33 kDa including QVH sequence; 61% sequence identity to Shewanella sp. UCD-KL21 accession WP 076541099.1)], two PL-17 enzymes [(80 kDa; 85% sequence identity to Aliivibrio logei accession WP 065609674.1) and (85 kDa; 79% sequence identity to Shewanella frigidimarina accession WP 081430169.1)] and one unclassified enzyme (50 kDa; 76% sequence identity to Aliivibrio wodanis accession WP 074050300.1). We will characterize the recombinant forms of these enzymes (produced in E. coli cells) in future studies to elucidate the mechanisms underlying the degradation of alginate by Shewanella sp. YH1 enzymes. Enzyme activities of AlgSI-PL7 Enzyme activity assays were completed using various buffers (Fig. 2A–C). We observed that AlgSI-PL7 was more active in phosphate buffer than in Tris buffer, which was in contrast to the results of a previous study, in which AlgC-PL7 activity was the same in both buffers (data not shown). However, other studies revealed that Tris buffer decreased or completely inhibited the activities of alginate lyases derived from Pseudoalteromonas sp. strain No. 272 and Turbo cornutus (45, 46). Although the reason for this buffer effect is still unclear, the amino group of Tris might adversely affect the stability of AlgSI-PL7 via electrostatic interactions. The fact that AlgSI-PL7 was highly active in the presence of 0.2 m NaCl or KCl indicates AlgSI-PL7 functions optimally at seawater salt concentrations. Additionally, AlgSI-PL7 retained approximately 50% of the activity at 1.0 m NaCl and KCl. Furthermore, acidic amino acids (Asp and Glu) can affect salt tolerance. We determined that about 13% of the AlgSI-PL7 amino acids are acidic. The acidic amino acid contents of salt-tolerant alginate lyases generally exceed 14%, while non-salt-tolerant alginate lyases contain approximately 11% acidic amino acids. Therefore, AlgSI-PL7 likely exhibits at least some salt tolerance (33). Moreover, the other PL-7 enzymes of Shewanella sp. YH1 also contain more than 13% acidic amino acids. However, the salt-tolerance of these enzymes will need to be experimentally verified. Effects of metal ions and inhibitors on AlgSI-PL7 The effects of metals on the activities of many alginate lyases have been examined (26). However, these effects are influenced by the environmental conditions (47). The effects of metals differ among enzymes, and there may be differences between polyG and polyM lyases. The polyG lyases tend to be activated by monovalent cations, and are inactivated by divalent cations. In contrast, the polyM lyases are activated by divalent cations. Details regarding the underlying mechanisms remain unclear, but a previous study observed that cations decreased the surface density of the substrate charge to weaken the ionic interaction between alginate and the enzyme (48). The local structures of the enzyme may be affected by cations. In this study, AlgSI-PL7 was inactivated by EDTA and N-bromosuccinimide. Earlier studies compared the differences in the effects of inhibitors between the polyG and polyM lyases (35, 49). Several M block-degrading enzymes were reportedly slightly activated or unaffected by chelators, while many enzymes that prefer G blocks are inactivated by EDTA. Our observed effects of EDTA on AlgSI-PL7 are consistent with those of previous studies because AlgSI-PL7 is a polyG lyase. It is also suggested that AlgSI-PL7 may harboured metal ions in its structure. Furthermore, the addition of N-bromosuccinimide, which is a tryptophan-modification reagent, completely inactivated the enzyme. Although AlgSI-PL7 has five tryptophan residues, two of them (positions 199 and 302) are located close to the catalytic region. Thus, the reagent may affect AlgSI-PL7 activity. Two cysteine residues are located in the C terminal region of AlgSI-PL7. Therefore N-ethylmaleimide and DTNB did not affect enzyme activity. Stability of AlgSI-PL7 We observed that AlgSI-PL7 exhibits unique thermostability characteristics (Fig. 2D). An incubation at 40°C caused AlgSI-PL7 to form aggregates and precipitate. In contrast, AlgSI-PL7 incubated at 70°C did not aggregate and remained soluble. Thus, establishing the correlation between the temperature-dependent protein conformation and the amino acid sequence is important. The crystal structures of the PL-7 enzymes have been well studied (50, 51). These enzymes generally have a jelly-roll fold, and CD data suggest that PL-7 enzymes are β-sheet structures. We observed that AlgSI-PL7 forms a β-sheet structure (Fig. 4), which is consistent with the findings of earlier studies (46, 52). The PL-7 enzymes generally contain 15 β-strands, with β-5, β-7 and β-14 representing conserved sequences (51). An abundance of β-strand structures is associated with the formation of β-aggregates and amyloid fibrils. Moreover, a partially unfolded protein with an exposed hydrophobic region tends to form amyloids (53–55). The PASTA algorithm (http://protein.bio.unipd.it/pasta2/) can be used to predict the amyloidogenic regions of a protein based on the amino acid sequence (56, 57). The results of PASTA analyses indicated that AlgSI-PL7 has a highly amyloidogenic region in the β-4 and β-7 strands. Interestingly, the β-7 strand is important for substrate recognition (and includes the QIH sequence). Thus, the β-7 strand of PL-7 alginate lyases is located at the substrate-binding area in the cleft of a globe-like conformation (51). When AlgSI-PL7 was incubated at 40°C for 1 h, the partial unfolding of the enzyme exposed the β-7 strand region, resulting in the formation of aggregates due to intermolecular interactions. The hydrogen bonds that form between intermolecular β-strands make it difficult for the enzyme to refold. However, at 70°C, AlgSI-PL7 is essentially completely unfolded (Fig. 4B). Unfolded proteins exhibit increased solubility and decreased protein–protein interactions, which may help to explain why AlgSI-PL7 incubated at 70°C did not form aggregates and was able to refold to its native state. These hypotheses are supported by the results of the ThT-binding assay in which increasing fluorescence corresponded to the formation of β-aggregates (Fig. 4A). Moreover, the same phenomena were observed for the rAlgSI-PL7 denatured by 8 m urea (completely unfolded) (Fig. 3B), which was also able to refold to its functional state. Many alginate lyases do not recover their activities immediately after heat or chemical treatments. Moreover, a previous analysis of secondary structures indicated the catalytic domain of AlgC-PL7 differs from that of other alginate lyases (33). Thus, AlgC-PL7 can refold, but the reconstruction of the catalytic domain takes time. Crystal structure analyses have been conducted to resolve this issue. We also completed a PASTA analysis to compare the tendencies of the other Shewanella sp. YH1 PL-7 lyases to aggregate. Although we did not detect any amyloidogenic regions in the β-7 strand, one PL-7 lyase, which contained the QVH sequence, had a highly amyloidogenic region in a non-catalytic area. Mechanism mediating the degradation of AlgSI-PL7 Several properties common among the PL-7 lyases have been confirmed by statistical and structural analyses. These properties suggest the enzymes generally have three conserved amino acid sequences that influence substrate recognition (30, 35, 58). In particular, the PL-7 lyases have two important amino acid sequences (QVH and QIH) that are thought to dictate substrate specificity (i.e. QVH: polyM-specific; QIH: polyG- and polyMG-specific). Our results suggest that AlgSI-PL7 exhibits high specific activity against polyG and low specific activity against polyM (Fig. 5). Additionally, AlgSI-PL7 contains a QIH sequence, implying it is a polyG lyase. Shewanella sp. YH1 also produces two QVH-lyases. The combined functions of these enzymes may contribute to the efficient degradation of alginate to oligoalginate. The degradation of alginate by AlgSI-PL7 results in the production of di-, tri-, tetra- and pentasaccharides, suggesting AlgSI-PL7 is an endo-type alginate lyase. Moreover, disaccharides are the main degradation products generated by AlgSI-PL7. These oligosaccharides can effectively induce the proliferation of keratinocytes in the presence of epidermal growth factor (59). In particular, disaccharides are believed to be the ideal unit for antioxidants, necessitating the production of abundant disaccharides (60). However, disaccharide yields are usually low among the products of most alginate lyases (61–63). An alginate lyase derived from Agarivorans sp. L11 appears to be a relatively rare exception, as it produces a high disaccharide yield (64). Therefore, continuing the search for new alginate lyases that can produce an abundant supply of disaccharides is critical. In conclusion, AlgSI-PL7 represents a potentially valuable enzyme for producing disaccharides. Moreover, its ability to refold to its active state suggests AlgSI-PL7 might be useful as a commercial enzyme. Supplementary Data Supplementary Data are available at JB Online. Acknowledgements We thank Dr Hirokazu Suzuki (Tottori University) for useful discussions. This work was supported by Grants for Scientific Research [15K18490 (2015–2016) and 17K07308 (2017–2020)] from the Ministry of Education, Science, Sports, and Culture, Japan. We also received financial assistance from the Takeda Science Foundation and the Uehara Memorial Foundation. We thank Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript. Author Contributions: H.Y. and T.O. designed the study. H.Y., A.F., and N.I. conducted the experiments. H.Y. and A.F. analysed the data. 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( 2013) Purification and characterization of a new alginate lyase from a marine bacterium Vibrio sp. Biotechnol. Lett.  35, 703– 708 Google Scholar CrossRef Search ADS PubMed  64 Li S., Yang X., Bao M., Wu Y., Yu W., Han F. ( 2015) Family 13 carbohydrate-binding module of alginate lyase from Agarivorans sp. L11 enhances its catalytic efficiency and thermostability, and alters its substrate preference and product distribution. FEMS Microbiol. Lett . 362 Abbreviations Abbreviations CD circular dichroism ThT thioflavin T © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)
TI - Characterization of a novel endo-type alginate lyase derived from Shewanella sp. YH1
JF - The Journal of Biochemistry
DO - 10.1093/jb/mvy001
DA - 2018-04-01
UR - https://www.deepdyve.com/lp/oxford-university-press/characterization-of-a-novel-endo-type-alginate-lyase-derived-from-aH8zHXKilZ
SP - 341
EP - 350
VL - 163
IS - 4
DP - DeepDyve
ER -