ORIGINAL RESEARCH PAPER

Purification and characterization of a thermostable k-carrageenase from a hot spring bacterium, Bacillus sp.

Jiang Li • Qiushi Hu • Dewi Seswita-Zilda

Received: 20 December 2013 / Accepted: 20 March 2014

� 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-carrageen-

ase was isolated from a hot spring in Indonesia and

identified as a Bacillus sp. The k-carrageenase, Cga-

L50, 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

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-carrageen-

ase from hot spring bacteria.

Keywords Bacillus � Carrageenase �Characterization � Hot spring bacteria � Neo-k-

carrabiose

Introduction

Carrageenans are gel-forming, linear sulfated-galac-

tans extracted from certain marine red algae. They

consist of D-galactose residues with alternating a-1,3-

and 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-D-

galactose), i-(2-sulfate-3,6-anhydro-a-D-galactopyr-

anosyl-1,4-4-sulfate-b-D-galactose), and k-carrageenan

(2,6-sulfate-a-D-galactopyranosyl-1,4-2-sulfate-b-D-

galactose) (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

J. Li (&)

Key Lab of Marine Bioactive Substances, The First

Institute of Oceanography, SOA, Qingdao 266061, China

e-mail: lijiang@fio.org.cn

Q. Hu

College of Chemical Engineering, Qingdao University of

Science & Technology, Qingdao 266061, China

e-mail: xiaonan96@qq.com

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: seswitazilda@yahoo.com

123

Biotechnol Lett

DOI 10.1007/s10529-014-1520-7

(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 oligosac-

charides (COS) exhibit valuable pharmacological

activities (Ren et al. 2010; Wang et al. 2011; Yuan

et al. 2006); in particular, k-carrabiose oligosaccha-

rides, 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 microorgan-

isms has now progressed to a considerable degree

owing to the potential applications of their thermo-

stable enzymes in various fields of biotechnology

(Huber and Stetter 1998; Ladenstein and Antranikian

1988). In the present study, a thermostable k-carrag-

eenase, named as Cga-L50, was purified from a hot-

spring bacterium, Bacillus sp. Lc50-1, and character-

ized. This enzyme, which could effectively degrade k-

carrageenan 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-carraoligo-

saccharide standards (purity: about 95 %) were kindly

provided by Dr. Feng Han and Dr. Xia Zhao, the

Ocean University of China.

Table 1 Summary of the Cga-L50 purification procedure

Steps Total

activity (U)

Total

protein (mg)

Specific

activity (U/mg)

Purification

(fold)

Recovery

(%)

Culture supernatant 1990 711 2.8 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

Sephacryl S-200HR 96 0.9 105.9 37.8 4.8

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

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

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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�3501200; 5�4404600). 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 k-

carrageenan as the sole energy and carbon source.

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

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;

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

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123

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, i-carrageenan, 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.

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 car-

rageenovora were stable only below 30 �C (Min 2008;

Guibet et al. 2007). Thermostability is considered as

an important and useful criterion for industrial

application of carrageenase. Thus, the high thermo-

tolerance 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-carra-

geenan, 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-

Fig. 3 TLC analysis of the oligosaccharides released from k-

carrageenan 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-carra-

pentaose 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

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Fig. 4 HPLC analysis of the oligosaccharides released from k-

carrageenan 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

Biotechnol Lett

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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-carrabi-

ose (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 carbo-

hydrate 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).

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