Biotechnol Lett DOI 10.1007/s10529-014-1520-7

ORIGINAL RESEARCH PAPER

Purification and characterization of a thermostable kcarrageenase from a hot spring bacterium, Bacillus sp. Jiang Li • Qiushi Hu • Dewi Seswita-Zilda

Received: 20 December 2013 / Accepted: 20 March 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Purpose of work The purpose of this study is to report a thermostable k-carrageenase that can degrade k-carrageenan yielding neo-k-carrabiose at 75 °C. A thermophilic strain Lc50-1 producing k-carrageenase was isolated from a hot spring in Indonesia and identified as a Bacillus sp. The k-carrageenase, CgaL50, with an apparent molecular weight of 37 kDa and a specific activity of 105 U/mg was purified from the culture supernatant. The optimum pH and temperature of Cga-L50 were 8.0 and 75 °C, respectively. The enzyme was stable from pH 6–9 and retained *50 % activity after holding at 85 °C for 10 min. Significant activation of Cga-L50 was observed with K?, Ca2?, Co2?, and Na?; whereas, the enzyme activity was inhibited by Sr2?, Mn2?, Fe2?, Cu2?,Cd2?, Mg2?, and

J. Li (&) Key Lab of Marine Bioactive Substances, The First Institute of Oceanography, SOA, Qingdao 266061, China e-mail: [email protected] Q. Hu College of Chemical Engineering, Qingdao University of Science & Technology, Qingdao 266061, China e-mail: [email protected] D. Seswita-Zilda Research Center for Marine and Fisheries Product Processing and Biotechnology, Agency for Marine and Fisheries Research and Development, Ministry of Marine and Fisheries Affairs, Jakarta 40115, Indonesia e-mail: [email protected]

EDTA. Cga-L50 is an endo-type k-carrageenase that hydrolyzes b-1,4-linkages of k-carrageenan, yielding neo-k-carrabiose as the main product. This study is the first to present evidence of thermostable k-carrageenase from hot spring bacteria. Keywords Bacillus  Carrageenase  Characterization  Hot spring bacteria  Neo-kcarrabiose

Introduction Carrageenans are gel-forming, linear sulfated-galactans extracted from certain marine red algae. They consist of D-galactose residues with alternating a-1,3and b-1,4-linkages. Based on the number and position of sulfate substitutions, as well as the presence of a 3,6-anhydro bridge in a-l,4-linked galactose residues, carrageenans are mainly classified into three types: j(3,6-anhydro-a-D-galactopyranosyl-1,4-4-sulfate-b-Dgalactose), i-(2-sulfate-3,6-anhydro-a-D-galactopyranosyl-1,4-4-sulfate-b-D-galactose), and k-carrageenan (2,6-sulfate-a-D-galactopyranosyl-1,4-2-sulfate-b-Dgalactose) (Campo et al. 2009). Enzymes that degrade different carrageenans are classified into j-, i-, and k-carrageenases, and belong to different glycoside hydrolase (GH) families listed in the carbohydrate-active enzymes (CAZy) database

123

Biotechnol Lett

(Cantarel et al. 2009). To date, j- and i-carrageenases have been widely studied, whereas there are only a few reports on k-carrageenases (Guibet et al. 2007; Min 2008; Ohta and Hatada 2006). Carrageenan oligosaccharides (COS) exhibit valuable pharmacological activities (Ren et al. 2010; Wang et al. 2011; Yuan et al. 2006); in particular, k-carrabiose oligosaccharides, containing 1,4-linked i-carrageenases -galactose 2,6-disulfate units and highly sulfated groups, possess biological activities with potential applications as new drugs. Therefore, detection of novel k-carrageenases with high activity has become the focus of research and development of new drugs. Carrageenan, at high concentrations, is highly viscous at room temperature and is an inhibitor of carrageenase degradation. The most commonly used method for obtaining low-viscous carrageen is to increase the solution temperature; however, most carrageenases are not stable above 40 °C (Zhou et al. 2008). Therefore, it is important to identify carrageenases with high thermostability that could play a significant role in the industrial applications of carrageenan. Research on thermophilic microorganisms has now progressed to a considerable degree owing to the potential applications of their thermostable enzymes in various fields of biotechnology (Huber and Stetter 1998; Ladenstein and Antranikian 1988). In the present study, a thermostable k-carrageenase, named as Cga-L50, was purified from a hotspring bacterium, Bacillus sp. Lc50-1, and characterized. This enzyme, which could effectively degrade kcarrageenan yielding neo-k-carradiaose at 75 °C, has the potential for use in industrial applications.

Materials and methods Materials The j-, i-, and k-carrageenans were purchased from Sigma. The carraoligosaccharide and neo-carraoligosaccharide standards (purity: about 95 %) were kindly provided by Dr. Feng Han and Dr. Xia Zhao, the Ocean University of China.

Fig. 1 SDS-PAGE of the purified Cga-L50. SDS-PAGE was performed with a stacking gel (4 % polyacrylamide) and a separating gel (10 % polyacrylamide) to estimate protein purity and molecular weight. Gels were stained with Fast Silver Stain Kit (Beyotime Institute of Biotechnology). Lanes 2 4 and 6 Standard molecular weight markers; lanes 1 and 3 Cga-L50 purified with Q-Sepharose FF column by different times; lane 5 Cga-L50 purified by Sephacryl S-200HR column after Q-Sepharose FF column

Table 1 Summary of the Cga-L50 purification procedure Steps

Total activity (U)

Total protein (mg)

Culture supernatant

1990

711

Specific activity (U/mg) 2.8

Purification (fold)

Recovery (%)

1

100

(NH4)2SO4 precipitation

537

59

9.1

3.2

27

Q-Sepharose F.F

138

2.4

56.6

20.2

6.9

96

0.9

105.9

37.8

4.8

Sephacryl S-200HR

Values given are the average of three replications. Strain Lc50-1 was grown in 500 ml vials containing 200 ml selective medium at 55 °C and 120 rpm for 24 h. The culture supernatant was obtained by centrifugation at 6,0009g for 30 min, and then precipitated with 70 % saturation with ammonium sulfate by slow stirring for 1 h. The clear dialysate was applied onto a Q-Sepharose column (2.6 9 40 cm) equilibrated with phosphate buffer (pH 7.5) and eluted (1 ml/min) by using a discontinuous gradient of NaCl (0–0.5 M) in the same buffer. The fractions with the highest k-carrageenase activity were pooled and dialyzed against 50 mM phosphate buffer (pH 7.5). The resulting solution was loaded onto Sephacryl S-200 column equilibrated with phosphate buffer (pH 7.5) and the proteins were eluted with the same buffer. All the steps were carried out at 4 °C

123

Biotechnol Lett

Isolation and identification of the bacteria The k-carrageenase-producing thermophilic bacterial strains were isolated from mud samples collected from the hot spring on the coast of Kalianda Island, Indonesia (105°350 1200 ; 5°440 4600 ). The strains were inoculated in broths containing 3 g peptone/l, 3 g yeast extract/l, 3 g NaCl/l, and 20 g agar/l, and incubated at 55 °C. Positive colonies showing clear zones were picked out from the selection plates containing kcarrageenan as the sole energy and carbon source.

Fig. 2 Characterization of the purified Cga-L50. a The optimal temperature of Cga-L50 was determined by measuring the enzyme activity in 50 mM sodium phosphate buffer (pH 7.5) at various temperatures (55–85 °C). b The optimal pH of Cga-L50 was determined by measuring the enzyme activity at 75 °C in 50 mM sodium acetate/acetic acid buffer (pH 3.8–6.0; squares), KH2PO4/NaOH buffer (pH 6–7.5; circles), Tris/HCl buffer (pH 7.5–9.0; triangles), and NaCO3/NaHCO3 buffer (pH 9–10;

The 16S rRNA sequence of the strain Lc50-1 was amplified by PCR from the genomic DNA and sequenced. The sequence was blasted and aligned with closely related sequences retrieved from GenBank using the BLASTn and CLUSTAL X program, respectively. Assay of k-carrageenase activity and substrate specificities Cga-L50, 100 ll, was incubated with 900 ll 0.2 % (w/v) substrate in 50 mM phosphate buffer (pH 7.5) at

stars). c Thermostability of the purified Cga-L50. The enzyme was incubated at 55 °C (squares), 65 °C (circles), 75 °C (triangles), and 85 °C (stars) for various time periods, and the residual activities were determined at 75 °C. d The effect of various ions or chelators on the catalytic activity of Cga-L50 was determined by including metal salts (2 mM) in the assay mixture and incubating at 75 °C for 15 min. The control comprised the assay mixture without metal ion salts or chelator

123

Biotechnol Lett

75 °C for 15 min. The reducing oligosaccharide products in the reaction mixture were assayed by using the 3,5-dinitrosalicylic acid method. One unit of carrageenase activity was defined as the amount of enzyme required to liberate 1 lmol reducing sugar per minute under the above-mentioned conditions. For the analyses of substrate specificities, j-carrageenan, icarrageenan, k-carrageenan, and agar were used as the substrate, respectively. Analysis of the degradation products A total of 0.5 ml (15 U/ml) of the purified enzyme was incubated with 2 ml k-carrageenan (2 g/l in 50 mM phosphate buffer; pH 7.5) for 15 min, 120 min and overnight at 75 °C, and subsequently analyzed by using TLC and HPLC.

application of carrageenase. Thus, the high thermotolerance of Cga-L50 allows its potential use in reactions with high concentrations of carrageenan at high solution temperature. The effects of metal ions and chelating agent on the activity of Cga-L50 are shown in Fig. 2d. Substrate specificity and degradation products Cga-L50 effectively degraded k-carrageenan but showed no activity towards j-carrageenan, i-carrageenan, and agar (data not shown). The main product formed after k-carrageenan hydrolysis by Cga-L50 was neo-k-carrabiose analyzed using TLC (Fig. 3). The product was also analyzed using HPLC with the retention time showing good agreement with that of neo-k-carrabiose standard (Fig. 4). Both of results suggest that Cga-L50 efficiently hydrolyzes b-1,4-

Results and discussion Isolation and identification of the strain Lc50-1 Of eight isolates, Lc50-1 exhibited the highest carrageenase activity and was selected for further examination. Analysis of the 16S rRNA sequences revealed that it belonged to the genus Bacillus, and hence, was named Bacillus sp. Lc50-1. Purification of Cga-L50 The typical purification procedure is summarized in Table 1. The purified Cga-L50 produced a single band on the SDS-PAGE gel, which showed an apparent molecular weight of 37 kDa (Fig. 1). Characterization of Cga-L5 Cga-L50 was most active at 75 °C and pH 8.0 in 50 mM Tris/HCl buffer (Fig. 2a, b), and was stable over a broad pH range (6–9) and retained 70 % of its original activity after incubation at 75 °C for 15 min (Fig. 2b). Furthermore, the enzyme retained 50 % of its activity when incubated at 75 °C for 45 min or 85 °C for 10 min (Fig. 2c). k-Carrageenases from Cellulophaga sp. QY20 and Pseudoalteromonas carrageenovora were stable only below 30 °C (Min 2008; Guibet et al. 2007). Thermostability is considered as an important and useful criterion for industrial

123

Fig. 3 TLC analysis of the oligosaccharides released from kcarrageenan by Cga-L50. Purified enzyme, 0.5 ml, (15 U/ml) was incubated with 2 ml k-carrageenan (2 g/l in 50 mM phosphate buffer; pH 7.5) 15 min, 120 min and overnight at 75 °C. The reaction products were separated on a TLC plate with n-butanol/acetic acid/water (2:2:1, by vol) and color developed. lane M1 standard mixture, j-carratriose, j-carrapentaose and j-carraheptaose; lane 1 reaction products for 15 min; lane 2 reaction products for 120 min; lane 3 reaction products overnight; lane M2 standard mixture, neo-k-carrabiose and neo-k-carratetraose

Biotechnol Lett

Fig. 4 HPLC analysis of the oligosaccharides released from kcarrageenan by Cga-L50. A total of 0.5 ml (15 U/ml) of the purified enzyme was incubated with 2 ml k-carrageenan (2 g/l in 50 mM phosphate buffer; pH 7.5) for 15 min, 120 min, and overnight at 75 °C, and the reaction products were precipitated with ethanol. The reaction products were analyzed by using HPLC on a Shodex OHpak SB-802.5 HQ (7.8 9 300 mm) with

0.1 M NaSO4 as an eluent, equipped with a refractive index detector. a Standard neo-k-carratetraose, with a retention time of 43.6 min. b Standard neo-k-carrabiose, with a retention time of 45.1 min. c Reaction products in 0 min. d Reaction products obtained in 15 min, with a retention time of 44.9 min. e Reaction products obtained in 120 min, with a retention time of 44.9 min

123

Biotechnol Lett

linkages of k-carrageenan yielding neo-k-carrabiose as the main product. This finding is different from that reported in earlier studies. The main final products of k-carrageenase from Cellulophaga sp. QY20 were reported to be neo-k-carratetraose and neo-k-carrabiose (Min 2008), while that of k-carrageenase from P. carrageenovora was neo-k-carratetraose (Guibet et al. 2007). Sulfated oligosaccharides from marine algae have diverse biological and physiological activities, which depend on structural parameters such as carbohydrate structure, molecular mass, degree of sulfate esterification, and the linking position of sulfo groups (Liu et al. 2000). Therefore, degraded carrageenans, and in particular k-carrabiose oligosaccharides, have great applicative potential in industry. Acknowledgments This study was financially supported by Public Science and Technology Research Funds Project of Ocean (201205024, 201405015).

References Campo VL, Kawano DF, Silva DB Jr et al (2009) Carrageenans: biological properties, chemical modifications and structural analysis—a review. Carbohydr Polym 77:167–180 Cantarel BL, Coutinho PM, Rancurel C et al (2009) The carbohydrate-active enzymes database (CAZy): an expert resource for glycogenomics. Nucleic Acid Res 37:233–238 Guibet M, Colin S, Barbeyron T et al (2007) Degradation of lambda-carrageenan by Pseudoalteromonas carrageenovora lambda-carrageenase: a new family of glycoside

123

hydrolases unrelated to kappa- and iota-carrageenases. Biochem J 404:105–114 Huber H, Stetter KO (1998) Hyperthermophiles and their possible potential in biotechnology. J Biotechnol 64:39–52 Ladenstein R, Antranikian G (1988) Advances in biochemical engineering. Biotechnology 61:37–85 Liu JM, Haroun-Bouhedja F, Boisson-Vidal C (2000) Analysis of the in vitro inhibition of mammary adenocarcinoma cell adhesion by sulphated polysaccharides. Anticancer Res 20:3265–3271 Liu GL, Li Y, Chi Z et al (2011) Purification and characterization of j-carrageenase from the marine bacterium Pseudoalteromonas porphyrae for hydrolysis of j-carrageenan. Proc Biochem 46:265–271 Min N (2008) Study on k-carrageenase of marine bacterium Cellulophaga sp. QY201. Ocean University of China, Qingdao Ohta Y, Hatada Y (2006) A novel enzyme, lambda-carrageenase, isolated from a deep-sea bacterium. Biochem J 140:475–481 Ren S, Li J, Wang W et al (2010) Protective effects of kappa-ca3000 ? CP against ultraviolet-induced damage in HaCaT and MEF cells. J Photochem Photobiol B 101:22–30 Sun FX, Ma YX, Wang Y et al (2010) Purification and characterization of novel j-carrageenase from marine Tamlana sp. HC4. Chin J Oceanol Limnol 28:1139–1145 Wang W, Zhang P, Hao C et al (2011) In vitro inhibitory effect of carrageenan oligosaccharide on influenza a H1N1 virus. Antivir Res 92:237–246 Yuan H, Song J, Li X et al (2006) Immunomodulation and antitumor activity of j-carrageenan oligosaccharides. Cancer Lett 243:228–234 Zhou MH, Ma JS, Li J et al (2008) A kappa-carrageenase from a newly isolated Pseudoalteromonas-like bacterium, WZUC10. Biotechnol Bioproc Eng 13:545–551