LI Shangyong,JIA Panpan,WANG Linna,YU Wengong,and HAN Feng

Key Laboratory of Marine Drugs, Chinese Ministry of Education; Provincial Key Laboratory of Glycoscience and Glycotechnology of Shandong Province and School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003,P.R.China

1 Introduction

Carrageenans are sulfated galactans consisting of D-galactose residues linked by alternating α-1,3 and β-1,4 glycosidic bonds,which are commercially important components of the cell walls of red seaweeds (Liuet al.,2010).The three most industrially exploited carrageenans,namely κ-,ι- and λ-carrageenans,are distinguishable each other; they contain one,two and three ester-sulfate groups per repeating disaccharide unit,respectively (Guibetet al.,2007).Carrageenans are widely used in industry owing to their unique physicochemical properties.The κ-carrageen-derived sulfated oligosaccharides have been reported to have anti-viral and anti-tumor (Mouet al.,2003),antiin fl ammation,antioxidant and immunolore- gulation activities (Yuanet al.,2006).

The κ-carrageenases (EC 3.2.1.83) cleave the internal β-1,4 linkages of κ-carrageenans,yielding oligogalactans of either neocarrabiose or neoagarobiose series.Several κ-carrageenases have been found in marine bacteria such asPseudoalteromonascarrageenovora(Collénet al.,2009),Pseudomonaselongata(Khambhatyet al.,2007),Zobelliagalactanovorans(Potinet al.,1991) andVibriosp.(Toshiyoshiet al.,1999).The genes of the κ-carrageenases have been cloned fromPseudoalteromonas carrageenovora(Barbeyronaet al.,1994) andCytophaga drobachiensis(Barbeyronet al.,1998).The κ-carrageen at high concentrations is highly viscous at room temperature and an inhibitor of κ-carrageenase degradation.The most commonly used method of obtaining low viscous κ-carrageen is to increase solution temperature; unfortunately most reported κ-carrageenases are not stable at temperartures above 40℃ (Zhouet al.,2008).

In this study,a new κ-carrageenase,namely CgkP,was purified from a marine bacteriumPseudoalteromonassp.QY203 and characterized.CgkP was relatively thermostable,promising for industrial applications.

2 Materials and Methods

2.1 Bacterial Strains,Media and Culture Conditions

The strain QY203 was isolated from the decayed red algae collected from the coast of Qingdao,China.It was cultured in a fermentation medium (30 g L-1NaCl,3 g L-1MgSO4·7H2O,0.2 g L-1CaCl2,0.1 g L-1KCl,0.02 g L-1FeSO4,3 g L-1Casein,1.5 g L-1Na2HPO4,1 g L-1NaH2PO4and 2 g L-1κ-Carrageenan) at 25℃ for 48 h in a shaker(150 r min-1).Escherichia coliDH5α was used as the host of recombinant plasmids.E.colicells were grown at 37℃in Luria-Bertani (LB) broth or on LB agar supplemented with (100 μg ampicillin) mL-1if necessary.

2.2 Molecular Identification of Strain QY203

Identi fi cation of strain QY203 was accomplished using the 16S rRNA gene (rDNA) sequencing.Briefly,the 16S rDNA was amplified from genomic DNA by PCR using the bacterial specific primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’) with a GeneAmp 9700 PCR system.The PCR product was inserted into pMD18-T vector (Sangon Biotech,China) and sequenced with a BigDye terminator sequencing method.Sequence analysis was performed by comparing the DNA sequences with those retrieved from GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi).The multiple sequence alignments were performed using ClustalX 1.83 with the phylogenetic trees constructed with MEGA 4.0 (Tamuraet al.,2007).

2.3 Purification of CgkP

Enzyme purification was always carried out at 4℃.The bacterial culture was centrifuged at 6000×g for 15 min.Into the supernatant,ammonium sulfate was added to a final saturation of 40%.After standing for 2 h,200 mL of the mixture was loaded onto a Phenyl-Sepharose column (1.6 cm×20 cm) equilibrated with 20 mmol L-1phosphate buffer (pH 7.2),then eluted with a linear gradient of(NH4)2SO4(1.5–0 mol L-1,100 mL) at a flow rate of 1 mLmin-1.The fractions (25 mL) showing κ-carrageenase activity was loaded onto a column of HiTrap Q (1.6 cm×10 cm) equilibrated with 20 mmol L-1phosphate buffer (pH 7.2).After washing,a linear NaCl gradient(0–1 mol L-1,100 mL) was superimposed on the starting buffer at 1 mL min-1.The active fractions were stored at?20℃.Protein concentration was determined by referring to bovine serum albumin (BSA) standard following Bradford method.Sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) was performed as the described (Laemmliet al.,1973).

2.4 Assay of CgkP Activity

Unless stated otherwise,0.9 mL of κ-carrageenan (2 g L-1in 20 mmol L-1sodium phosphate buffer,pH 7.2) was incubated with 0.1 mL of the enzyme at 45℃ for 15 min.The reaction was stopped by heating at 100℃ for 10 min.The reducing sugar released was quantified with dinitrosalicylic acid (DNS) reagent (Miller,1959).One unit of κ-carrageenase activity was defined as the amount of enzyme releasing 1 μmol reducing sugar (measured as galactose) from κ-carrageenan per minute.

2.5 Characterization of the Purified CgkP

The optimal acidity of the enzyme was determined by using 50 mmol L-1Na2HPO4-citric acid (pH 4.0–7.0),50 mmol L-1Na2HPO4-NaH2PO4(pH 6.6–8.6),100 mmol L-1Tris-HCl (pH 7.6–9.0) and 50 mmol L-1Gly-NaOH (pH 9.0–11.0) buffers in the assay system.To determine acidity stability,the residual activity was measured after enzyme was incubated in above buffers (pH 4.0–11.0) at 4℃ for 6 h.The optimal temperature of the enzyme was determined by measuring the activity at various temperatures (10–60℃).The thermostability of the enzyme was determined by measuring the residual activity after the enzymes in 20 mmol L-1phosphate buffer (pH 7.2) were incubated at 35℃,40℃,45℃ and 50℃,respectively,for various times.

2.6 Mode of κ-Carrageenan Degradation by CgkP

Mixtures of 5 mL CgkP (2 U mL-1) and 50 mL κ-carrageenan (2 g L-1in 20 mmol L-1phosphate buffer,pH 7.2)were incubated at 45℃ for up to 30 min.An aliquot of hydrolysis product (0.5 mL) was taken out at different times (1,5,10,15 and 30 min) in order to determine the viscosity and reducing sugar.Viscosity was measured as described previously by Kobayashiet al.(2009).

2.7 Analysis of CgkP Degrading Products

Five milliliters of purified enzyme (5 U mL) was incubated with 20 mL of κ-carrageenan (2 g L-1in 20 mmol L-1phosphate buffer,pH 7.2) at 37℃ overnight.Thin-layer chromatography (TLC) analysis was performed on a HPTLC plates (Merck,Germany) with a solvent of nbutanol/acetic acid/water (2:1:1) (Toshiyoshiet al.,1999).The end products of κ-carrageenase degrading reaction were fractionated on a Biogel-P6 column (1.6 cm ×100 cm).Elution was performed with 0.2 mol L-1NH4HCO3at a flow rate of 0.05 mL min-1.

3 Results and Discussion

3.1 Identification of Strain QY203

Sixty-two colonies (strains) were isolated,of them,39 produced κ-carrageenase and 15 showed high κ-carrageenase activity (>6 U mL-1) after being cultured at 25℃for 48 h.Of the 15 strains with high enzyme activity,strain QY203 showed the highest κ-carrageenase activity(12 U mL-1).The 16S rDNA of strain QY203 (GenBank accession number JQ669941) was 1430 bp in length.BLASTn search revealed that the highest similarity of 99.4% existed between the strain QY203 andPseudoalteromonassp.P102.The phylogenetic tree (Fig.1) shows that the strain QY203 belongs to genusPseudoalteromonas.Strain QY203 is a species in genusPseudoalteromonas,which was designated asPseudoalteromonassp.QY203.

3.2 Purification and Properties of CgkP

The enzyme was purified 88.3 folds through (NH4)2-SO4precipitation,hydrophobic chromatography and anionic-exchange chromatography (Table 1).The specific activity and yield were 1121.7 U mg-1and 26.9%,respectively.

The purity of the purified CgkP was confirmed by SDS-PAGE (Fig.2).A single protein band was stained,which was estimated to be 34.0 kDa in molecular weight.and the Mws of the κ-carrageenases from the genusPseudoalteromonaswas at least 40.0 kDa.For example,Mw of κ-carrageenase fromPseudoalteromonas porphyraeLL1 was 40.0 kDa (Liuet al.,2010),Mw of κ-carrageenase CgkA fromPseudoalteromonas carrageenovora(ATCC 43555) was 44.4 kDa (Barbeyronet al.,1994) andPseudoalteromonas elongatayielded κ-carrageenase with a Mw of 128.0 kDa (Khambhatyet al.,2007).

Fig.1 Phylogenetic tree of strain QY203 and related bacteria based on a maximum parsimony analysis of 16S rDNA sequences.Number after the strain names are culture collection numbers followed by GeneBank accession numbers of 16S rDNA sequences.

Table 1 Summary of CgkP purification

Fig.2 SDS-PAGE of CgkP.Lane M,molecular weight markers,lane 1,puri fi ed CgkP.The gel was stained with Coomassie Brilliant Blue.

The activity of CgkP in phosphate buffer was the highest at pH 7.2 (Fig.3A) and stable within a range of pH 6.0–9.0 (Fig.3B).The optimal pH value of CgkP was different from that of κ-carrageenases purified fromPseudoalteromonas porphyraeLL1 andPseudoalteromonas carrageenovoraATCC 43555 (pH 8.0) (Liuet al.,2010).The optimal temperature of CgkP was 45℃ (Fig.3C).CgkP remained 95% and 80% of activity after incubation at 40℃ and 45℃ for 1 h,respectively (Fig.3D).CgkP still retained 70% activity after being incubated at 40℃ for 48 h.It retained 40% and 20% activity even being incubated at 50℃ for 1 h and 3 h,respectively.The κ-carrageenases fromP.porphyrae,P.carrageenovoraandCytophaga drobachiensiswere stable only at temperatures below 30℃ (Liuet al.,2011).The κ-carrageenases fromCytophagasp.MCA-2 andPseudoalteromonassp.WZUC10 were stable at 40℃ for less than 2 h (Zhouet al.,2008).Although the κ-carrageenase fromTamlanasp.HC4 was stable at temperatures below 45℃ for 2 h,only 10% activity was retained after being incubated at 50℃ for 30 min (Sunet al.,2010).In comparison,CgkP was thermostable,promising for industrial application.

CgkP was active without NaCl; however,its activity was enhanced markedly by NaCl.The activity of CgkP was increased by 240% by 300 mmol L-1NaCl (Table 2).Its activity was also enhanced by K+.In comtrast,divalent and trivalent metal ions including Cu2+,Ni2+,Zn2+,Mn2+,Al3+and Fe3+significantly inhibited the activity of CgkP;while Mg2+was not effective on its activity.The chelating agent EDTA inhibited the activity of CgkP,suggesting that it was a metalloenzyme (Bernardoet al.,2004).Detergent SDS reduced the activity of CgkP,which was similar to most κ-carrageenases reported previously.

Fig.3 Effects of acidity and temperature on the activity and stability of CgkP.(A) The optimal acidity of CgkP.Buffers were 50 mmol L-1 Na2HPO4-citric acid (◇),50 mmol L-1 Na2HPO4-NaH2PO4 (●),100 mmol L-1 Tris-HCl (△) and 50 mmol L-1 Gly-NaOH (◆).(B) The acidity stability of CgkP.The same buffers were used as above.(C) The optimal temperature of CgkP.(D) The thermostability of CgkP.CgkP was incubated at 35℃ (◇),40℃ (●),45℃ (△) and 50℃ (■) for up to 48 h.

Table 2 Effect of metal ions,chelators and detergents on κ-carrageenase activity of CgkP

3.3 Analysis of Degradation Products

After adding CgkP,the viscosity of κ-carrageenan solution decreased rapidly in first 5 min,but changed little in following 25 min.The amount of reducing sugar (A520)increased steadily in 30 min,the whole observation period(Fig.4).These findings suggested that the enzyme was an endo-κ-carrageenase characterized by Kobayashiet al.(2009).

Fig.4 Decrease of κ-carrageenan viscosity during enzymatic degradation.Filled circles with a solid line,the rate of viscosity; open circles with a dotted line,the absorbance at 520 nm.

The end degrading products of κ-carrageenan by CgkP were disaccharide and tetrasaccharide as were characterized with TLC analysis (Fig.5).Out the Biogel-P6 column,most eluted hydrolysates formed two distinct peaks which were identified as κ-carrageenan-derived neocarradiaose and neocarratetraose with electrospray ionization mass spectrometry (Data not shown).It has been documented that κ-carrageenases fromPseudomonasandVibriodegrade κ-carrageenan into disaccharide and tetrasaccharide (Liuet al.,2010); while κ-carrageenase fromPseudoalteromonas carrageenovoraATCC43555 degrades κ-carrageenan into neocarradiaose,neocarratetraose and neocarrahexraose (Barbeyronet al.,1994).

Fig.5 TLC analysis of the oligosaccharides derived from κ-carrageenan after incubation with CgkP.Lane 1,control,κ-carrageenan with inactivated CgkP; lane 2,end products of κ-carrageenan hydrolyzed by CgkP; lane 3,κ-neocarratetraose sulfate; lane 4,κ-neocarrabiose sulfate; lane 5,galactose.

4 Conclusion

An endo-κ-carrageenase,CgkP,was purified fromPseudoalteromonassp.QY203 with a simple and reliable method.It is a new thermostable κ-carrageenase promising for industrial use.

Acknowledgements

This work was supported by National Science Foundation of China (31000361 and 31070712),Program for Changjiang Scholars and Innovative Research Team in University (IRT0944),Special Fund for Marine Scientific Research in the Public Interest (201005024) and the Fundamental Research Funds for the Central Universities(201013008).

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