WANG Linna, LI Shangyong, ZHANG Shilong, LI Jiejing, YU Wengong, and GONG Qianhong

Key Laboratory of Marine Drugs of Chinese Ministry of Education,Shandong Provincial Key Laboratory of Glycoscience & Glycotechnology,School of Medicine and Pharmacy,Ocean University of China,Qingdao266003,P. R. China

A New κ-Carrageenase CgkS from Marine Bacterium Shewanella sp. Kz7

WANG Linna, LI Shangyong, ZHANG Shilong, LI Jiejing, YU Wengong, and GONG Qianhong*

Key Laboratory of Marine Drugs of Chinese Ministry of Education,Shandong Provincial Key Laboratory of Glycoscience & Glycotechnology,School of Medicine and Pharmacy,Ocean University of China,Qingdao266003,P. R. China

A new κ-carrageenase genecgkSwas cloned from marine bacteriumShewanellasp. Kz7 by using degenerate and site-finding PCR. The gene was comprised of an open reading frame of 1224 bp, encoding 407 amino acid residues, with a signal peptide of 24 residues. Based on the deduced amino acid sequence, the κ-carrageenase CgkS was classified into the Glycoside Hydrolase family 16. ThecgkSgene was expressed inEscherichia coli, and the recombinant enzyme was purified to homogeneity with a specific activity of 716.8 U mg-1and a yield of 69%. Recombinant CgkS was most active at 45℃ and pH 8.0. It was stable at pH 6.0-9.0 and below 30℃. The enzyme did not require NaCl for activity, although its activity was enhanced by NaCl. CgkS degraded κ-carrageenan in an endo-fashion releasing tetrasaccharides and disaccharides as main hydrolysis products.

κ-Carrageenan; cloning; characterization; oligosaccharide;Shewanella

1 Introduction

Carrageenans are linear sulfated galactans extracted from red seaweeds and share a common backbone of D-galactose with alternating α (1-3) and β (1-4) linkages (Yaoet al., 2013). Depending on the presence of a 3,6-anhydro bridge in the β-l,4-linked galactose residue and on the position and number of sulfate substituents, they are referred to as κ-, ι-, or λ-carrageenans (Campoet al., 2009).

Three types of hydrolases, which degrade κ-, ι-, and λ-carrageenans at β-l,4-linkages are named as κ-, ι-, and λ-carrageenases respectively, and belong to different glycoside hydrolase (GH) families in the carbohydrate-active enzymes (CAZy) database (Cantarelet al., 2009). κ-Carrageenases belongs to GH family 16, and cleave κ-carrageenans yielding oligogalactans of the neocarrabiose series. The κ-carrageenan-derived sulfated oligosaccharides have been reported to have anti-viral, anti-tumor anti-inflammation, anti-oxidant and immunolo-regulation activities (Mouet al., 2003; Yuanet al., 2006). κ-Carrageenase could be used as a powerful tool to prepare specific κ-carrageenan oligosaccharides for further study on biological activity-structure relationship and industrial exploitation (Sunet al., 2010). Most of κ-carrageenaseswhich have been characterized were purified from marine wild type bacteria strains (Potinet al., 1991; Barbeyronet al., 1994; Liet al., 2013). The application of natural κ-carrageenases has been limited by complex purification procedures and low yield. Heterologous expression is an efficient way of enhancing enzyme production and the recombinant enzymes can be purified by one-step affinity chromatography with a high yield. But very few recombinant κ-carrageenases have been studied. The recombinant Cgk-K142a fromPseudoalteromonas tetraodonisJAM-K142 has showed very low activity (Kobayashiet al., 2012). The end products of κ-carrageenan hydrolyzed by recombinant CgkZ fromZobelliasp. ZM-2 are complex mixtures and hard to separate (Liuet al., 2013). Therefore, it is essential and important to find new recombinant κ-carrageenase suitable for industrial production and purification of oligosaccharides.

Here, we cloned and expressed a new κ-carrageenase CgkS fromShewanellasp. Kz7. It degraded κ-carrageenan, yielding κ-carrageenan tetrasaccharides and disaccharides as the main products with a high specific activity of 716.8 U mg-1.

2 Materials and Methods

2.1 Strain and Oligonucleotides

Shewanellasp. Kz7 was isolated from sea mud collected along the coastal zone of Jiaozhou Bay, Qingdao,China, and preserved in China Center for Type Culture Collection (CCTCC) under the accession number AB 2014040. Oligonucleotides used for the gene cloning and expression of CgkS are shown in Table 1.

2.2 Cloning of the κ-Carrageenase Gene and Sequence Analysis

Degenerate primers (CgkS-F, CgkS-R) were designed according to the conserved sequences of GH family 16 κ-carrageenases to amplify the partial sequence of κ-carrageenase gene. A 690-bp DNA fragment was obtained and sequenced. The flanking sequences were obtained using the SiteFinding-PCR method (Tanet al., 2005) with nine nested specific primers (SFP1&2&3, Down-CgkS-sf-sF1&2&3, Up-CgkS-sf-asF1&2&3). The signal peptide was predicted using SignalP 4.0 server (http：//www. cbs.dtu.dk/ services/SignalP). Theoretical molecular weight and isoelectric point (pI) were then calculated using Compute pI/Mw tool (http：//us.expasy.org/tools/pi_tool. html).

2.3 Expression and Purification of Recombinant CgkS

For expression of His-tagged CgkS, the DNA fragment containingcgkSgene without signal sequence and stop codon was amplified using the primers (CgkS-EF and CgkS-ER), and then ligated into theNdeI andXhoI sites of expression plasmid pET28a (Novagen, USA). The resulting expression plasmid pET28-cgkS was transformed into the expression strainE.coliBL21 (DE3). Protein expression was induced at OD600of 0.8 with 0.5 mmol L-1isopropyl-β-thiogalactoside (IPTG) for 36 h at 25℃ and 100 r min-1in LB medium containing 30 μg kanamycin mL-1. Cells were harvested and disrupted by sonication, and then cell debris and unbroken cell were removed by centrifuge. The recombinant CgkS was purified from the soluble fraction using a Ni-Sepharose column. The purity and molecular weight of purified CgkS were determined by SDS-PAGE on a 10% resolving gel.

2.4 κ-Carrageenase Activity Assay and Protein Determination

κ-Carrageenase activity was measured by using the 3,5-dinitrosalicylic acid (DNS) method. The enzymatic hydrolysis reaction was conducted in 20 mmolL-1phosphate buffer (pH 8.0) containing 0.2% (w/v) κ-carrageenan (Sigma) at 45℃ for 10 min. One unit (1 U) of enzyme activity was defined as the amount of enzyme that released 1 μmol reducing sugar (measured as D-galactose) per minute under the above conditions. The protein concentration was measured using the method of Bradford with bovine serum albumin as the standard.

2.5 Determination of Kinetic Parameters

Initial velocities were determined in the standard assay mixture at 20 mmolL-1phosphate buffer (pH 8.0). The kinetic parameters of CgkS were measured by using ten different concentrations of κ-carrageenan (ranging from 0.1 to 5 mg mL-1). TheKmandVmaxwere then analyzed by using Lineweaver-Burk methods.

2.6 Analysis of Hydrolysis Product and Pattern of CgkS

The reaction mixture containing 0.5 mL (10 U) purified enzyme and 2 mL κ-carrageenan (2 g κ-carrageenan L-1) in 20 mmolL-1phosphate buffer (pH 8.0) was incubated overnight at 45℃, then the hydrolysis products were analyzed by thin layer chromatography (TLC) (Huet al., 2013). To determine the hydrolysis pattern, the reducing sugars were monitored by DNS method, and the relative viscosity was measured by a viscometer at time intervals as described previously by Kobayashiet al. (2009).

2.7 Nucleotide Sequence Accession Numbers

The nucleotide sequence forcgkSwas deposited in GenBank under the accession number KJ000056.

3 Results and Discussion

3.1 Cloning and Sequence Analysis of the κ-Carrageenase Gene

The κ-carrageenase gene,cgkS, consisted of an open reading frame of 1224 bp, encoding 407 amino acid residues, including a signal peptide of 24 residues. The molecular weight and pI of the mature enzyme deduced from its amino acid sequence were 42 743 Da and 9.1, respectively. CgkS had the highest identity of 70% with κ-carrageenase (Genbank ADD92366) fromPseudoalteromonassp. LL1, and had the identity of 68% with alkaline κ-carrageenase Cgk-K142a (Genbank AB572925) fromP. tetraodonisJAM-K142. Based on the catalytic domain (Glu163-Asp165-Glu168), the enzyme is a new member of GH family 16 (Liuet al., 2013).

3.2 Purification and Biochemical Characterization of CgkS

Fig.1 SDS-PAGE of CgkS. LaneM, molecular weight markers; Lane 1, purified CgkS.

The recombinant CgkS was purified to apparent homogeneity with a 69% yield by one-step affinity chro-matography, and migrated as a band of 45 kDa on SDSPAGE (Fig.1), which was in good agreement with the calculated molecular weight of fusion protein. The specific activity of the recombinant CgkS was 716.8 U mg-1, and much higher than those of the recombinant CgkZ (107.3 U mg-1) and Cgk-K142a (8.16 U mg-1). Although the natural κ-carrageenase CgkP shows a higher specific activity (1121.7 U mg-1), the application of CgkP has been limited by complex purification procedures and low yield (26.9%) (Liet al., 2013).

CgkS showed an apparentKm of 0.15 ± 0.04 mg mL-1and aVmax of 807.6 ± 82.6 U mg-1protein. The optimal temperature of CgkS was 45℃ (Fig.2a), and 85% of the enzymatic activities remained after being incubated at 30℃ for 1 h (Fig.2b). CgkS showed the highest activity in phosphate buffer at pH 8.0 (Fig.2c) and it was stable within a range of pH 6.0-9.0 (Fig.2d).

NaCl was not necessary for the enzymatic activity, though it enhanced the activity (Table 1). However, all of tested divalent and trivalent metal ions, such as Cu2+, Ni2+, Zn2+, Mg2+, Al3+, Fe3+, showed a significantly inhibitory effect except for Ca2+and Mn2+. The chelating agent EDTA slightly inhibited the activity of CgkS, suggesting that this enzyme is not a metalloenzyme (Bernardoet al., 2004). Detergent SDS reduced the activity of CgkS, which was the same with most κ-carrageenases reported previously. CgkS specifically hydrolyzed κ-carrageenan. No activity was observed on λ-, ι-carrageenan or agar (data not shown).

Table 1 Effect of metal ions, chelators and detergents on the activity of CgkS

Fig.2 Effects of pH and temperature on the activity and stability of CgkS. a) The optimal temperature of CgkS was determined by measuring the activity at various temperatures (20-70℃). b) The optimal pH of CgkS was determined measuring the activity at 45℃ in 50 mmol L-1Na2HPO4-citric acid (open rhombus), 50 mmol L-1Na2HPO4-NaH2PO4(filled circle), 100 mmol L-1Tris-HCl (open triangle) and 50 mmol L-1Gly-NaOH (filled rhombus). c) The thermostability of CgkS was studied by measuring the residual activity after the enzymes were incubated at different temperatures for 1 h in 20 mmol L-1phosphate buffer (pH 7.0). d) pH stability of CgkS. The residual activity was measured at 45℃ in 20 mmol L-1phosphate buffer (pH 8.0) after incubation from pH 4 to 9.6 with the above buffers for 6 h at 4℃. 100% activity= 22.6 U mL-1.

3.3 Analysis of Hydrolysis Product and Pattern

After completion of κ-carrageenan degradation by CgkS, the main products were tetrasaccharides and disaccharides by TLC analysis (Fig.3). Then the main products were purified by a Biogel-P6 column and analyzed by negative-ion electrospray ionization mass spectrometry (ESI-MS). The spectra (data not shown) showed good agreement with those of κ-carrageenan-derived neocarratetraose and neocarrahexraose, which was reported previously (Duanet al., 2010).

Fig.3 TLC analysis of the oligosaccharides derived from κ-carrageenan. CgkS, 0.5 mL, (20 U mL-1) was incubated with 2 mL κ-carrageenan (2 g L-1in 20 mmol L-1phosphate buffer, pH 8.0) overnight at 45℃. The reaction products were separated on a HPTLC plate withn-butanol/formic acid/water (2：1：1) and color-developed. Lane M, standard mixture, κ-neocarratetraose and κ-neocarrabiose; Lane 1, κ-carrageenan; Lane 2, reaction products of κ-car- rageenan hydrolyzed by CgkS.

Fig.4 Decrease of κ-carrageenan viscosity during enzymatic degradation. Mixtures of 5 mL CgkS (5 U mL-1) and 50 mL κ-carrageenan (2 g L-1in 20 mmol L-1phosphate buffer, pH 8.0) were incubated at 45℃ for up to 60 min. An aliquot of hydrolysis product (0.5 mL) was taken out at different times (1, 5, 10, 15, 30 and 60 min) in order to determine the viscosity and reduce the sugar. Filled circles with a solid line, the rate of viscosity; open circles with a dotted line, the absorbance at 520 nm.

The κ-carrageenan has also been degraded into disaccharide and tetrasaccharide by the natural κ-carrageenases which are purified from the genus ofPseudoalteromonas,PseudomonasandVibrio(Liuet al., 2010). However, the end products of κ-carrageenan hydrolyzed by recombinant CgkZ are tetrasaccharides, hexasaccharides, octasaccharides, and decasaccharides (Liuet al., 2013).

The hydrolysis pattern of CgkS was determined by viscometric assay. The viscosity rapidly decreased to 10% of the original during the first 5 min of incubation, and decreased slowly by only 5% during the later 55 min of incubation. However, the amount of reducing sugar (A520) increased steadily during the whole 60 min period (Fig.4). These results revealed that CgkS degraded κ-carrageenan in an endo-fashion.

4 Conclusion

The biological activities of κ-Carrageenan oligosaccharides are closely related with the degree of polymers. To prepare specific κ-carrageenan oligosaccharides for further structure-activity relationship study, it is essential to use suitable κ-carrageenases. The application of natural κ-carrageenases has been limited by complex purification procedures and low yield. The known recombinant κ-carrageenases are also not suitable for application because of low activity or complex mixture of degradation products (Liuet al., 2013; Kobayashiet al., 2012). The new recombinant κ-carrageenase CgkS exhibits a high specific activity to κ-carrageenan in the absence of NaCl, yielding κ-carrageenan-derived neocarratetraose and neocarrahexraose as the main products. Therefore, CgkS would play a significant role in further industial application of κ-carrageenan oligosaccharides.

Acknowledgements

The research was supported by the Key Technologies Research and Development Program of China (2013BA B01B02), National Science Foundation of China (310707 12), Special Fund for Marine Scientific Research in the Public Interest (201005024 and 201105027) and National Hightech R&D Program of China (2011AA09070304).

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(Edited by Ji Dechun)

(Received July 10, 2014; revised February 29, 2015; accepted March 21, 2015)

? Ocean University of China, Science Press and Springer-Verlag Berlin Heidelberg 2015

* Corresponding author. Tel： 0086-532-82032067 E-mail： gongqh@ouc.edu.cn