Electrospray Ionization Mass Spectrometric Analysis of κ

There are three main commercial carrageenans: kappa (κ), iota (ι), and ... For κ-carrageenan, the G-unit is β-d-galactose-4-sulfate (G4S) and the ...
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Electrospray Ionization Mass Spectrometric Analysis of κ‑Carrageenan Oligosaccharides Obtained by Degradation with κ‑Carrageenase from Pedobacter hainanensis Yujiao Sun, Yang Liu, Kuan Jiang, Chengjian Wang, Zhongfu Wang, and Linjuan Huang* Educational Ministry Key Laboratory of Resource Biology and Biotechnology in Western China, Life Science College, Northwest University, Xi’an 710069, P. R. China S Supporting Information *

ABSTRACT: κ-Carrageenan was degraded with a novel κ-carrageenase isolated from Pedobacter hainanensis, which was first isolated from seaside soil under the stacks of red algae in Hainan province of China. The κ-carrageenase was detected with a molecular weight of ∼55 kDa estimated from SDS−PAGE and yielded enzymatic activity of 700.53 units/mg of protein under the conditions of pH 7.0 and 40 °C. Analysis of the degradation products by TLC and HPLC indicated that the enzyme degraded κ-carrageenan to sulfated oligosaccharides with even-numbered degree of polymerization, of which the tetrasaccharide was the major product. All the degradation components during different time courses were analyzed by ESI-MS, and their structures were assigned. Structural analysis by CID MS/MS revealed that each carrageenan oligosaccharide was composed of An-G4S-type neocarrabiose units, which consisted of a 3,6-anhydro-α-D-galactose (An) residue in the nonreducing end and a βD-galactose-4-sulfate (G4S) residue in the reducing end. These results demonstrated that the κ-carrageenase cleaved κcarrageenan at the internal β-1,4 linkage of κ-carrageenan. This enzymatic degradation offers an alternative approach to prepare κ-carrageenan oligosaccharides, which could be used as a powerful tool for further study on biological activity−structure relationship and thorough industrial exploitation of κ-carrageenan. KEYWORDS: κ-carrageenase, κ-carrageenan oligosaccharides, TLC, HPLC, ESI-MS, CID MS/MS

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INTRODUCTION Carrageenans are a family of water-soluble linear sulfated galactans extracted from marine red algae consisting of alternating 1,3-linked β-D-galactopyranosyl (G) and 1,4-linked α-D-galactopyranosyl (D) units. According to the position and number of sulfate groups and the number of 3,6-anhydrogalactosyl rings of each disaccharide unit, carrageenans can be classified into different types. There are three main commercial carrageenans: kappa (κ), iota (ι), and lambda (λ)-carrageenans. For κ-carrageenan, the G-unit is β-D-galactose-4-sulfate (G4S) and the D-unit is 3,6-anhydro-α-D-galactose (An).1,2 Carrageenans are widely used in food, cosmetic, and biomedical industries. Moreover, the carrageenan oligosaccharides are potential pharmaceutical products with multiple biological activities including anticoagulant, antioxidant, anti-inflammatory, and antiviral activities.3−11 These biological activities are closely related with the position and the number of sulfate groups, as well as the degree of polymerization (Dp). However, it is still a challenge to elucidate a whole carrageenan polysaccharide molecule’s detailed sequence and linkage. Therefore, structural analysis of carrageenan oligosaccharides provides an opportunity to study the relationships between structures and biological activities of the self-associating polysaccharides.12−14 Thus, it has become increasingly important to produce and purify oligosaccharides from polysaccharides. Carrageenases are powerful tools for detailed structural analysis of carrageenans. The enzymes cleave carrageenans and lead to a rapid disruption of polysaccharides. The digestion © 2014 American Chemical Society

produces lower molecular weight oligosaccharides that exhibit relatively higher bioactivities than original polysaccharides.15−18 Carrageenases are highly specific to a given class of carrageenans. For example, κ-carrageenase only degrades κcarrageenan, while ι-carrageenase and λ-carrageenase work for ι-carrageenan and λ-carrageenan, respectively.19−22 Recently, several carrageenases have been isolated and purified from marine bacteria such as Alteromonas (or Pseudoalteromonas), Pseudomonas, and Cytophaga.23−27 These enzymes specifically hydrolyze the internal β-1,4 glycosidic linkage between 3,6anhydro-D-galactose and D-galactose, yielding a series of carrageenan oligosaccharides.1,28 However, many carrageenases have not been purified to homogeneity and are not very well characterized. None of these carrageenases is commercially available.2 Moreover, the degradation products remain to be characterized in detail.29 Recently, a new bacterial strain, designated as Pedobacter hainanensis 13-QT, was isolated from seaside soil in Hainan province of China for the first time.30 The fermentation product of this strain could degrade κ-carrageenan. However, the degradation process and hydrolysates remain to be investigated. The primary objective of this work was to unravel the structural information of hydrolysates and the dynamics of enzymatic hydrolysis. TLC and HPLC were used to monitor Received: Revised: Accepted: Published: 2398

October 17, 2013 February 21, 2014 February 26, 2014 February 26, 2014 dx.doi.org/10.1021/jf500429r | J. Agric. Food Chem. 2014, 62, 2398−2405

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DTT. The loading amount of protein samples was 10 μg. Electrophoresis was carried out at 80 V in stacking gel and 120 V in separating gel for 4 h using an electrophoretic buffer consisting of Trisglycine with 0.1% SDS (pH 8.3). The gels were stained with staining buffer containing 1% (w/v) Coomassie Blue-R250, 40% aqueous ethanol, and 10% aqueous acetic acid for 30 min and subsequently destained with 40% aqueous ethanol and 10% aqueous acetic acid. Visualization of the proteins was carried out using ImageQuant 350 (GE Healthcare, Piscataway, NJ, USA). Preparation of κ-Carrageenan Oligosaccharides. κ-Carrageenan was enzymatically digested with κ-carrageenase. A 10-unit aliquot of enzyme solution was added to 100 mL of 0.2% (w/v) κ-carrageenan in 0.05 mol/L Tris-HCl buffer at pH 7.0, and the solution was incubated at 40 °C. 5 mL of the sample was taken out at intervals (i.e., 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 16 h, 20 h, 24 h, 28 h, 32 h, 48 h, 72 h, and 96 h) and boiled in a 100 °C water bath to stop the reactions. The products were concentrated and desalted using gel-filtration chromatography on a Bio-Gel P-2 column (40 cm × 2 cm). The column was eluted with distilled water using a pump with a flow rate of 0.8 mL/min, and fractions of 4 mL were autocollected. The elution was monitored by the phenol−sulfuric acid method.35 Fractions containing sugar were pooled together and lyophilized for further analysis. TLC Analysis of κ-Carrageenan Hydrolysate. Each 2 μL aliquot of 2 mg/mL reaction mixture was loaded onto a 20 cm × 20 cm plastic TLC plate precoated with 0.2 mm silica gel (Macherey-Nagel, Düren, Germany) and developed with a solvent system consisting of nbutanol:formic acid:water (4:7:1, v/v/v) at 25 °C. The developed plate was stained by spraying with orcinol−sulfuric acid reagent (0.2 g of orcinol, 20 mL of 98% H2SO4, 100 mL of methanol) and heated at 150 °C until the bands were obviously visible.36 3-Amino-9-ethylcarbazole (AEC) Labeling and HPLC Analysis of κ-Carrageenan Hydrolysate. Five milligrams of κ-carrageenan oligosaccharides obtained at different intervals was dissolved in 500 μL of deionized water. Two hundred microliters of 0.2 mol/L AEC in methanol, 25 μL of 0.5 mol/L aqueous sodium cyanoborohydride (NaBH3CN), and 50 μL of acetic acid were added successively. The mixtures were incubated at 70 °C for 1 h.37 These labeling reactions were then terminated by neutralization with 100 μL of 1.0 mol/L NaOH. The aqueous phase was extracted with 500 μL of dichloromethane to remove excess AEC, and then centrifuged at 8000 rpm for 3 min. The aqueous phase was taken out for chromatographic analysis. HPLC separation was performed on a Shimadzu LC-2010AHT (Tokyo, Japan). The column used was a 250 mm × 4.6 mm i.d., 5 μm, Sinochrom ODS-BP, with a 4 mm × 4 mm i.d. guard column of the same material (Elite Analytical Instruments Co., Ltd., Dalian, China). Data were collected and processed through Shimadzu Labsolutions software, version 5.51. The labeled oligosaccharides were analyzed by UV detection at 254 nm. Injection volume was 10 μL. The separation was performed at 40 °C in a linear gradient mode from 20% to 40% acetonitrile in 10 mmol/L ammonium acetate aqueous solution (pH 4.5) over 60 min. The flow rate was 0.5 mL/ min. Preparation of κ-Carrageenan Oligosaccharide Alditols. A 0.5 mL aliquot of 4% (w/v) aqueous sodium borohydride (NaBH4) solution was added to 2 mg of the freeze-dried sample, and the mixture was incubated at 25 °C for 2 h.38 The reaction was terminated by neutralization with 50 μL of acetic acid to destroy excess NaBH4. Boric acid was removed by repeated coevaporation with 0.1% anhydrous methanol/HCl (v/v) three times. The oligosaccharide alditols were analyzed by CID MS/MS. ESI-MS and CID MS/MS Analysis. The κ-carrageenan oligosaccharides were analyzed using a LTQ-XL ion-trap mass spectrometer equipped with an electrospray ion source and a HPLC system (Thermo-Fisher, Waltham, MA, USA). Data acquisition and processing were performed through the Xcalibur software. Aqueous samples (1 mg/mL) were injected via a 2 μL Rheodyne loop, and taken into the electrospray ion source by a stream of 50% aqueous methanol at a flow rate of 200 μL/min from the pump of HPLC system. For the electrospray ion source, the spray voltage was set at 4 kV, with a sheath gas (nitrogen gas) flow rate of 30 arbitrary units, an

the process of enzymatic degradation. Negative-ion ESI-MS and CID MS/MS were employed for structural analysis and sequence of hydrolysates.

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MATERIALS AND METHODS

Materials. Purified food-grade κ-carrageenan was purchased from Lubao Biochemistry Co., Ltd. (Jinjiang, China). Pedobacter hainanensis 13-QT was a strain gifted from the laboratory of Dalian Institute of Chemical Physics (Dalian, China). 3-Amino-9-ethylcarbazole (AEC), sodium cyanoborohydride (NaBH3CN), and albumin of chicken egg white were purchased from Sigma-Aldrich (St. Louis, MO, USA). BioGel P-2 Gel (45−90 μm wet bead size) and Bio-Gel P4̅ Gel (90−180 μm wet bead size) were purchased from Bio-Rad Laboratories, Inc. (Hercules, USA). Molecular weight protein markers (PageRuler Unstained Protein ladder) were purchased from Fermentas Scientific Inc. (Vilnius, Lithuania). Water was purified by a Milli-Q ultrapure water purification system from Millipore (Burlington, MA, USA). All other chemicals used in the experiments were of analytical reagent grade. Methods. Bacterial Strains and Culture Conditions. 50 μL of Pedobacter hainanensis 13-QT bacteria glycerol stock was inoculated into 10 mL of medium containing the following: κ-carrageenan (5 g/ L), peptone (3 g/L), NaNO3 (1 g/L), NaCl (20 g/L), K2HP4·3H2O (1 g/L), MgSO4·7H2O (0.5 g/L), and CaCl2 (0.1 g/L), dissolved in 1 L of distilled water, pH 7.5, and then incubated at 30 °C on a rotary shaker at a speed of 200 rpm overnight.31 Cells were subcultured by transferring 200 μL of the cell culture into 40 mL of the abovedescribed culture medium prior to incubation on a rotary shaker overnight (200 rpm) at 30 °C. The bacterial cells were cultured and grown to OD600 of 0.6−1.0 to reach the maximum output of κcarrageenase. Purification of κ-Carrageenase. The culture medium (100 mL) was centrifuged at 8000 rpm for 30 min. Subsequently, 80% ammonium sulfate was added to the supernatant and left overnight. The precipitates were collected by centrifugation at 8000 rpm for 50 min and then dissolved in 5 mL of 0.05 mol/L Tris-HCl buffer (pH 7.0). The clear supernatant containing κ-carrageenase was collected by centrifugation at 5000 rpm for 30 min and evaluated for enzymatic activity, where its protein content was estimated by the Bradford method.32 Then the purified κ-carrageenase was applied to a Bio-Gel P4̅ column (120 cm × 2 cm), equilibrated in 0.05 mol/L Tris-HCl buffer (pH 7.0), and eluted with the same buffer. Fractions were collected by continuous monitoring of the activity of κ-carrageenase. The pooled fraction was assayed for its total enzymatic activity and protein content. In order to protect the activity of κ-carrageenase, all the aforesaid steps were carried out at 4 °C. κ-Carrageenase Assays. The determination of enzymatic activity was assayed by measuring the increase in the concentration of reducing sugar ends formed by enzymatic digestion, using the dinitrosalicylic method of Miller.33,34 Boiled enzyme was used as a blank. 100 μL of the enzyme solution was added to 900 μL of 0.2% (w/v) κ-carrageenan in 0.05 mol/L Tris-HCl buffer (pH 7.0), and the mixture was incubated at 40 °C for 20 min. After incubation, 750 μL of modified 3,5-dinitrosalicylic acid reagent was added to the solution, and the mixture was heated for 5 min in a boiling water bath and then cooled in ice water. When the sample was cooled, 5 mL of deionized water was added and the absorption at 520 nm was measured in a (NewCentury-T6) spectrophotometer (Purkinje General Instrument Co., Ltd., Beijing, China). The amount of released sugar was estimated using a D-galactose calibration curve (0.2 to 0.8 mg/mL), and one unit of κ-carrageenase activity was defined as the amount of enzyme that produces the equivalent of 1 μmol of D-galactose from κ-carrageenan per minute under the above-described condition. Molecular Weight Determination. The molecular weight determination of κ-carrageenase was analyzed using SDS−PAGE with a 5% (w/v) polyacrylamide for the stacking gel, a 10% (w/v) polyacrylamide for the running gel, and 0.1% (w/v) SDS. One milligram of enzyme was denatured by heating at 100 °C for 10 min in 200 μL of protein denaturation solution containing 5% SDS and 6.2% 2399

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auxiliary gas (nitrogen gas) flow rate of 5 arbitrary units, a tube lens voltage of −250 V, a capillary temperature of 350 °C, and a capillary voltage of −48 V. Scan rate was normal, and the type was full, with a microscan number of 3, searching for a mass range of m/z 200−2000. When CID MS/MS was carried out, helium (He) was used as the collision gas, and the collision energy was set at 25−30% with an isotope width of m/z 3.00.

after the four steps of purification, the κ-carrageenase activity increased from 5.72 units/mg of protein to 700.53 units/mg of protein, leading to a 122.47-fold purification from the crude culture supernatant. The degree of purification is shown in Table 1. With references to the reported data, it can be concluded that the purified enzyme has much higher activity than those of some enzymes from other different sources. Khambhaty et al.24 have reported the purification of κcarrageenase from Pseudomonas elongate, exhibiting maximum enzymatic activity of 426.19 units/mg of protein. Sarwar et al.39 have reported a characterized κ-carrageenase from Cytophaga spp. having enzymatic activity of 5.0 units/mg of protein. Molecular Weight of κ-Carrageenase. The molecular weight of κ-carrageenase was determined by SDS−PAGE. As presented in Figure 1, the protein band of κ-carrageenase appeared as a single one between 50 kDa and 60 kDa standard molecular weight markers. The results indicated that the κcarrageenase has an apparent molecular weight of ∼55 kDa. TLC Analysis of κ-Carrageenan Oligosaccharides Hydrolysis by κ-Carrageenase. TLC analysis is a potent method to detect various oligosaccharides of different polymerization degrees.40 The presence of sulfate groups in sulfate oligosaccharides has an effect on migration and resolution of these oligosaccharides on TLC plates.41 With molecular mass information obtained by ESI-MS analysis, the κ-carrageenan oligosaccharides were observed and monitored on TLC plates (Figure 2). Lanes a, b, c, and d,

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RESULTS AND DISCUSSION Purification of κ-Carrageenase. After incubation, the cells were removed by centrifugation and 100 mL of crude culture supernatant was obtained. The original κ-carrageenase activity was 5.72 units/mg protein, which was set as a 1-fold purification. The proteins were then precipitated by ammonium sulfate, resulting in enzymatic activity of 60.46 units/mg of protein with a 10.57-fold purification from the crude culture supernatant. Afterward, the proteins were redissolved in TrisHCl buffer and concentrated to 5 mL of solution. The enzymatic activity reached to 107.25 units/mg protein with a 18.75-fold purification. The proteins were further purified on a Bio-Gel P4̅ column, which separated two peaks (B and C) of proteins with κ-carrageenase activity. Peak B, containing higher κ-carrageenase activity, was combined, and peak C was omitted due to a lesser amount of protein and lower activity. Furthermore, peak B was concentrated and the solution was applied for rechromatography on Bio-Gel P4̅. According to the rechromatographic pattern, peak B was found to have only one active peak. It was further confirmed by SDS−PAGE where only a single protein band was observed (Figure 1). Overall,

Figure 2. TLC analysis of κ-carrageenan oligosaccharides prepared through κ-carrageenase at pH 7.0 and 40 °C of a−n represented for samples of 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 16 h, 20 h, 24 h, 28 h, 32 h, 48 h, 72 h, and 96 h, respectively. Figure 1. SDS−PAGE analysis of κ-carrageenase purified from Pedobacter hainanensis 13-QT.

the product mixtures mainly containing oligosaccharides with various polymerization degrees between disaccharide (Dp 2) and decasaccharide (Dp 10), were observed as distinctive

Table 1. Purification of κ-Carrageenase from Pedobacter hainanensis no.

step

total vol (mL)

1 2 3 4

crude enzyme (NH4)2SO4 precipitation Tris-HCl redissolution gel filtration

100 40 5 3

total act.a (units) 184.30 356.11 14.05 13.31

± ± ± ±

1.96 2.86 0.25 0.14

total proteina (mg)

sp act.b (units/mg)

purifnc (-fold)

32.22 ± 0.46 5.89 ± 0.18 0.131 ± 0.0044 0.019 ± 0.0078

5.72 60.46 107.25 700.53

1 10.57 18.75 122.47

Values are expressed as means ± SD (n = 3). bSpecific activity = average of total activity/average of total protein. cPurification fold = specific activity/5.72 (units/mg); the value of 5.72 was original specific activity, designed as a 1-fold purification. a

2400

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separated bands on the TLC plate. After hydrolysis for 12 h, the spots of oligosaccharides at Dp 10 and higher polymerization degrees became faint, indicating that κ-carrageenan was effectively degraded. The reaction mixtures loaded on lanes i, j, k, l, m, and n corresponding to 24−96 h digestion mainly contained oligosaccharides ranging from disaccharide to hexasaccharide, of which tetrasaccharide was the dominant component. HPLC Analysis of κ-Carrageenan Oligosaccharides Hydrolysis by κ-Carrageenase. To quantitately assess the yield of oligosaccharides of different Dps, each sample was labeled by reductive amination with AEC and analyzed by HPLC. Some chromatographic data of the reaction products of enzymatic hydrolysis for 0.5 h, 12 h, 24 h, and 96 h are depicted in Figure 3. The peaks corresponding to Dp 2, Dp 4, Dp 6, and Figure 4. The yield curve of Dp 2, Dp 4, Dp 6, and Dp 8 obtained with different time courses analyzed by HPLC.

performed in negative-ion mode.42 The mass spectra of the oligosaccharide mixtures obtained at 12 h, 24 h, and 96 h are depicted in Figure 5. The mass spectra exhibited signals of multiply charged molecular anions, including singly, doubly, and triply charged

Figure 3. HPLC analysis of κ-carrageenan oligosaccharides obtained by κ-carrageenase at pH 7.0 and 40 °C (A) for 0.5 h; (B) for 12 h; (C) for 24 h; (D) for 96 h.

Dp 8 were clearly detected. Over 96 h, the area of the peaks of higher molecular weight fractions gradually decreased. The quantitative data of Dp 2, Dp 4, Dp 6, and Dp 8 are summarized in Figure 4. Obviously, typical oligosaccharides were obtained at different time courses. According to the peak area of these oligosaccharides, Dp 4 was the dominant component. The content of Dp 4 was low at the beginning, higher in the middle, and lower at the end of the reaction, and the highest yield of Dp 4 was about 70% of total oligosaccharides until 24 h. The curve of Dp 6 was similar to that of Dp 4. In contrast, the area curve of Dp 8 was in a quite different trend. The content of Dp 8 reached the highest point in the beginning and then decreased continuously to the end. During the process of enzymatic hydrolysis, the content of Dp 2 gradually increased. However, Dp 2 had a relatively low yield. ESI-MS Analysis of κ-Carrageenan Oligosaccharides Hydrolysis by κ-Carrageenase. Due to the presence of sulfate groups on the oligosaccharides, all MS experiments were

Figure 5. The ESI-MS analysis of κ-carrageenan oligosaccharides prepared by κ-carrageenase at pH 7.0 and 40 °C (A) for 12 h; (B) for 24 h; (C) for 96 h. 2401

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Figure 6. The CID MS/MS analysis of κ-carrageenan oligosaccharides and oligosaccharide alditols degraded by enzymatic hydrolysis. (A1) disaccharide vs (A2) disaccharide alditol, and (B1) tetrasaccharide vs (B2) tetrasaccharide alditol. Note the different fragments present in each spectrum, arising from the different linkages.

G4S)3H]2−. The oversulfated disaccharide units of AnG2S-G4S are attributed to inclusion of ι-carrageenan, a common impurity in commercial κ-carrageenan.42,43 The peak found at m/z 527.08 corresponds to the triply charged octasaccharide anion [(An-G4S)4Na]3−. The peak found at m/z 955.00 corresponds to doubly charged desulfated decasaccharide anions [(AnG4S)4(An-G)2Na]2−. The κ-carrageenan oligosaccharides obtained by digestion using κ-carrageenase for 24 h are mainly between Dp 2 and Dp 6, and their MS profile is depicted in Figure 5B. We found that the peaks at m/z 811.00 and m/z 789.00 correspond to molecular anions as sodium salt [(An-G4S)2Na]− and hydrogen salt [(An-G4S)2H]−, respectively. In the mass spectra of hexasaccharide, the base peak at m/z 1219.25 corresponds to the molecular anion with two sodium atoms ([(AnG4S)32Na]−); while the peaks found at m/z 391.25 and

ones. As shown in Figure 5A, the sample prepared in 12 h contained a series of κ-carrageenan oligosaccharides, including disaccharide, tetrasaccharide, hexasaccharide, octasaccharide, and decasaccharide. The peak at m/z 403.08 is assigned to disaccharide [(G4S-An)]− carrying one sulfate group. The peaks corresponding to molecular anion as sodium salt [(G4SAn)2Na]− (m/z 811.08) and potassium salt [(G4S-An)2K]− (m/z 827.00) are assigned to tetrasaccharide carrying two sulfate groups. The base peak found at m/z 1218.92 is likely to be representative of hexasaccharide with three sulfate groups bearing two sodium ions [(G4S-An)32Na]−. The peak at m/z 547.08 corresponds to doubly charged desulfated hexasaccharide anions [(An-G4S)2(An-G)]2− carrying two sulfate groups. This desulfation could be formed in the mass spectrometer due to high cone voltage.42 The peak found at m/z 667.00 is assigned to the doubly charged anion [(AnG2S-G4S)2(An2402

dx.doi.org/10.1021/jf500429r | J. Agric. Food Chem. 2014, 62, 2398−2405

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598.25 correspond to triply and doubly charged anions [(AnG4S)3]3− and [(An-G4S)3Na]2−, respectively. After enzymatic digestion for 96 h, tetrasaccharide was the major product, as shown in Figure 5C. The peak at m/z 394.17 corresponds to doubly charged tetrasaccharide anion [(AnG4S)2]2−. These results showed enzymatic properties of the κcarrageenase, for which the cleavage group was An-G4S unit, resulting in a series of neocarrabiose oligosaccharides with the code of An-G4S. Sequencing of κ-Carrageenan Oligosaccharides by CID MS/MS. Glycosidic cleavages provide information on constituent monosaccharide sequence and branching, whereas cross-ring cleavages can be used to define linkages. The crossring cleavage ions are generally less abundant when compared with the glycosidic cleavage ions.44−46 A nomenclature for describing these fragment ions was proposed by Domon and Costello47 and widely accepted. The detailed structures of κ-carrageenan oligosaccharides obtained by enzymatic hydrolysis were elucidated by CID MS/ MS. In order to differentiate fragment ions with identical masses arising from glycosidic cleavages, the oligosaccharide mixture was reduced by NaBH4 to obtain oligosaccharide alditols. Due to the reduction, the reducing terminal fragment ions showed an increase of 2 Da in CID MS/MS analysis. Therefore, the difference of reducing and nonreducing terminal fragments can be easily identified from each other.48,49 In the obtained MS profiles, all the oligosaccharide molecular ions showed 2 Da increase after the reduction treatment (data not shown). During the CID MS/MS analysis, disaccharide (m/z 403.25) and its alditol (m/z 405.08) form, together with tetrasaccharide (m/z 811.25) and its alditol (m/z 813.00) form, were taken as the precursors to determine the structure of κcarrageenan oligosaccharides. The CID MS/MS spectra are depicted in Figure 6, and the assignment data are shown in Table 2. In the MS/MS profiles of the disaccharide (Figure 6A1) and its alditol form (Figure 6A2), both glycosidic and cross-ring

fragments are found. The two major glycosidic fragment ions derived from the disaccharide alditol corresponding to the peaks at m/z 243.92 and 261.92 (Figure 6A2) are 2 Da increased from the peaks at m/z 241.92 and 259.75 (Figure 6A1). In the CID MS/MS spectra of the disaccharide and its alditol, Y- and Z-type glycosidic cleavage fragments are considerably more abundant than the cross-ring cleavage fragments. The cross-ring cleavage fragment ions at m/z 152.92, 199.75, 138.92, and 180.83 are derived from the sulfated disaccharide precursor ion. The ions at m/z 152.92, 180.83, and 138.92 are assigned to 3, 5A2-, 2, 5A2-, and 2, 4A2-type fragments, respectively. The peak at m/z 199.75 is assigned to 1, 3 X1-type. In the CID MS/MS spectra of tetrasaccharide (Figure 6B1) and its alditol (Figure 6B2), mainly glycosidic cleavage fragment ions as sodium adducts are observed but no crossring cleavage fragments are found in the spectra. Minor desulfation occurs, as indicated by the presence of weak signals at m/z 692.08, 694.08 (−120 Da, equivalent to the loss of NaHSO4), 708.08 (−103 Da, equivalent to the loss of NaSO3), and 734.08 (−80 Da, equivalent to the loss of SO3). The peaks at m/z 260.92 and 670.00 (Figure 6B2) show a 2 Da shift compared with the peaks at m/z 259.00 and 668.00 (Figure 6B1). These peaks are assigned to the reducing side fragments produced by glycosidic cleavages. The peaks at m/z 530.08 and 548.08 (Figure 6B2) observed here remain the same as the peaks at m/z 530.00 and 548.00 (Figure 6B1). These peaks are derived from cleavages at the nonreducing side of both tetrasaccharide and its alditol, determining that sulfated G4S are at the reducing terminal residue. Therefore, according to the spectral feature of glycosidic fragments from nonreducing (B- and C-type ions) and reducing sides (Y- and Z-type ions), the sequence of κ-carrageenan oligosaccharides prepared by κcarrageenase could be readily deduced as (An-G4S)n, which would give rise to oligosaccharides with terminal G4S residue. According to ESI-MS and MS/MS analysis, the enzymatic products were a homologous series of even-numbered sulfated oligosaccharides with (An-G4S)n, of which An is at the nonreducing end and G4S is at the reducing end. This result indicates that the κ-carrageenase of Pedobacter hainanensis cleaves at the internal β-1,4 linkages of κ-carrageenan (Figure 7).

Table 2. Fragment Ions Observed in the CID MS/MS Spectra of Disaccharide, Tetrasaccharide, and their Alditols as Precursors obsd fragment ion (m/z) of enzymic products (An-G4S)n and Their Alditols disaccharide fragment ion type

403

405

A2 3,5 A2 2,5 A2 1,3 X1 [(Z1-ene)]− Y1 [(Z2-ene)]− or [(B2-ene)]− C2 or Y2 [(B3-ene)]− C3 [(Z3-ene) + Na]− [Y3 + Na]− [M + Na]−/−NaHSO4 [M + Na]−/−NaSO3 [M + Na]−/−SO3

138.92 152.92 180.83 199.75 241.92 259.75

138.83 152.67 181.92 200.83 243.92 261.92

2,4

tetrasaccharide 811

241.00 259.00 385.00 403.00 530.00 548.00 649.92 668.00 692.08 708.08

813

260.92 385.00 402.92 530.08 548.08 670.00 694.08

Figure 7. Schematic diagram showing two disaccharide-repeating units of κ-carrageenan. The site of enzymatic hydrolysis is specific at the internal β-1,4 linkages of κ-carrageenan.

734.08 2403

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Journal of Agricultural and Food Chemistry

Article

The purified κ-carrageenase isolated from Pedobacter hainanensis is a novel enzyme acting as a β-hydrolyase to produce κ-carrageenan oligosaccharides with An-G4S-type, yielding tetrasaccharide as the main end product. The action pattern of this enzyme was quite different from those of other κ-carrageenases reported earlier. Ma et al.50 have reported a κcarrageenase isolated from Pseudoalteromonas sp. They indicated that the main end products were dimers of different neocarrabioses, e.g., disaccharide, tetrasaccharide, hexasaccharide, octasaccharide, and decasaccharide. Potin et al.26 have reported a κ-carrageenase purified and characterized from Cytophaga strain Dsji. They showed that tetrasaccharide and hexasaccharide were the major end products and the purified enzyme was not capable of hydrolyzing tetrasaccharide and hexasaccharide. Enzymatic activity, hydrolytic products, and various characteristics showed that the κ-carrageenase from Pedobacter hainanensis could be an alternative resource to produce better quality carrageenan with commercial value.

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ASSOCIATED CONTENT

S Supporting Information *

Figure S1: Gel filtration chromatography of κ-carrageenase on Bio-Gel P4̅ column. Figure S2: Rechromatography of the selected peak on Bio-Gel P4̅ column. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-029-88305853. Fax: +86-029-88303534. E-mail: [email protected]. Funding

This work was supported by the National Natural Science Foundation of China (NO. 21375103 and 31071506). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The help from Prof. Yuguang Du and Dr. Yanyu Meng in Dalian Institute of Chemical Physics (Chinese Academy of Sciences) in obtaining the strain of Pedobacter hainanensis 13QT is greatly acknowledged.

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ABBREVIATIONS USED Dp, degree of polymerization; CID MS/MS, electrospray tandem mass spectrometry with collision-induced dissociation; G, 1,3-linked β-D-galactopyranosyl; D, 1,4-linked α-D-galactopyranosyl; An, 3,6-anhydro-α-D-galactose; G4S, β-D-galactose4-sulfate; SDS−PAGE, sodium dodecylsulfate polyacrylamide gel eletrophoresis; SDS, sodium dodecylsulfate; DTT, dithiothreitol; AEC, 3-amino-9-ethylcarbazole

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