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Abstract Acid-solubilized collagen (ASC) and pepsin-solubilized collagen (PSC) were extracted from the seaweed pipefish (p箦諮. 鍉碥祀=肱茕芍射 and ...
Biotechnology and Bioprocess Engineering 2009, 14: 436-442 DOI/10.1007/s12257-009-0007-1

Isolation and Biochemical Characterization of Collagens from Seaweed Pipefish, Syngnathus Schlegeli Sher Bahadar Khan1†, Zhong-Ji Qian1†, BoMi Ryu2, and Se-Kwon Kim1,2* 1

Marine Bioprocess Research Center, Pukyong National University, Busan 608-737, Korea 2 Department of Chemistry, Pukyong National University, Busan 608-737, Korea

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^Äëíê~Åí= Acid-solubilized collagen (ASC) and pepsin-solubilized collagen (PSC) were extracted from the seaweed pipefish (póåÖJ å~íÜìë=ëÅÜäÉÖÉäá) and partially characterized. The amount of collagens isolated in the subsequent treatments was 5.5% of ASC and 33.2% PSC on the basis of lyophilized pipefish body weight, respectively. According to the electrophoretic pattern and CM-cellulose column chromatogram, the collagens might be classified as type I collagens, containing α1 and α2 chain. The imino acid content of collagen from pipefish was lower than those of mammalian collagens as also were the denaturation temperatures (Td) of collagens were 34.8°C and 35.1°C, respectively. This study shows that there is a possibility to use pipefish collagen as the alternative source of collagen from industrial purposes and subsequently it may evaluate the economical value of the seaweed pipefish. © KSBB hÉóïçêÇëW=Åçää~ÖÉåI=ëÉ~ïÉÉÇ=éáéÉÑáëÜI=áëçä~íáçåI=ÅÜ~ê~ÅíÉêáò~íáçå

INTRODUCTION Collagen exists in nearly all organs of vertebrates and is the major structural element of skin, bone, tendon, cartilage, blood vessels, and teeth. It is the most abundant and ubiquitous protein in the body of vertebrates [1]. In Medicine and Pharmacy, collagen has been regarded as one of the most useful biomaterials, mainly due to its biocompatibility, non-toxicity, and well-documented structural, physical, chemical, and immunological properties [2]. Collagen’s reported low antigenicity and immunogenicity has been related to the similarity in the amino acid sequence between species and to the modest content of aromatic amino acids [3]. Therefore, it has a wide range of applications in leather and film industries, pharmaceutical, cosmetic and biomedical materials, and foods [4-6], such as production of wound dressings, vitreous implants and carriers for drug delivery, edible casings [7], and production of cosmetics for good moisturizing properties [8]. In addition, collagen seems to play a significant nutritional role and its use in the meat industry as nutritive fibres or as a meat substitute has †

The first two authors equally contributed to this work.= G`çêêÉëéçåÇáåÖ=~ìíÜçê= Tel: +82-51-629-7094 Fax: +82-51-629-7099 e-mail: [email protected]

also been suggested [9]. However, for industrial purposes, the main sources of collagen are limited to those of land-based animals, such as bovine or porcine skin and bone. However, the outbreak of bovine spongiform encephalopathy (BSE), transmissible spongiform encephalopathy (TSE), and foot-andmouth disease (FMD) crisis have resulted in anxiety among users of collagen and collagen-derived products of land animal origin [10]. In addition, the collagen extracted from porcine cannot be used as a component of some foods due to religious barriers. Therefore, alternative sources of collagen should be sought. Scientists have found that skin, bone, scale, fin and cartilage of freshwater and marine fish, scallop mantle [11], the muscle layer of the ascidian [12], and adductor of pearl oyster [13] can be used as the new sources of collagen. Seaweed pipefish, Syngnathus schlegeli a marine teleost fish, is well known not only for its special medicinal composition and used as one of the most famous and expensive materials of Traditional Chinese medicine (TCM). It has proved to be particularly rich source of collagen so that pipefish has a potential, as an important source of collagens without the threat of BSE and TSE. In the present study, we isolated and characterized collagens extracted from pipefish of S. schlegeli. Furthermore, we compared the characterization of two collagens from pipefish to that of cattle or fish collagens by investigating biochemical and physical properties of collagens in order to use pipefish collagen as an alternative source of col-

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lagen from industrial purposes and subsequently it may increase the economical value of the seaweed pipefish.

MATERIALS AND METHODS j~íÉêá~äë

The live adults of seaweed pipefish were collected from Zhoushan Island, Zhejiang, China, in October 2005, and freeze-dried after removing the internal organs locus in quo. It was identified as seaweed pipefish S. schlegeli by professor Ming-Lu Den (Zoologist, Changchun University of Chinese Medicine, China). The pipefish were rapidly separated and rinsed with deionized water to eliminate contaminants under -4oC and then stored at -20oC until use. Pepsin (EC 3.4.23.1, 1:10,000) was obtained from Sigma Chemical Company (USA). ρ-dimethylaminobenzaldehyde, collagen type I (from porcine) and Achromopeptidase from Achomobacter llyticus (EC 3.4.21.50; 4.5 amidase activity/mg protein) were purchased from Wako Pure Chemicals industries (Tokyo, Japan). All other reagents used in this study were reagent grade chemicals and all procedures to isolate collagens were carried out at 4oC.

NaCl to a final concentration of 1.0 M. The precipitate was dissolved in 0.5 M acetic acid and dialyzed against 0.1 M acetic acid, and then lyophilized. The lyophilizates were named as PSC, which is pipefish pepsin-solubilized collagen. pçÇáìã=açÇÉÅóä=pìäÑ~íÉJéçäó~Åêóä~ãáÇÉ=dÉä= = bäÉÅíêçéÜçêÉëáë=EpapJm^dbF=

Protein patterns of collagen samples were analysed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli [14]. The collagen samples were dissolved in 0.02 M phosphate buffer (pH 7.2) containing 1% SDS and 3.5 M urea. The sample mixtures were gently stirred at 4oC for 12 h to dissolve total proteins. Supernatants were collected after centrifuging at 3,000g for 3 min at 4oC. Solubilised collagen samples were mixed with the sample buffer (0.5 M Tris-HCl, pH 6.8 containing 4% (w/v) SDS, 20% (v/v) glycerol) and using the sample/sample buffer ratio of 1:1 (v/v). Electrophoresis was performed on 6% gels in 0.1 M phosphate buffer (pH 7.2) containing 0.1% SDS. Each gel was visualized with Coomassie Brilliant Blue R250 dissolved in water, methanol, and trichloroacetic acid (5:4:1, v/v/v) and destained using a solution containing methanol, distilled water, and acetic acid (5:4:1, v/v/v).

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The collagens were extracted according to the method of Jongjareonarak et al. [10] with a slight modification. Seaweed pipefish was cut into small pieces with scissors. The pipefish was suspended in 10 volumes (w/v) of 0.1 M NaOH and the suspension was stirred overnight. Each solution was re-suspended in 20 volumes of 0.1 M NaOH solution with stirring for 24 h. The alkaline insoluble components were strained through cheesecloth and rinsed with distilled water repeatedly until a neutral pH was obtained. The insoluble matter was extracted with 10 volumes of 0.5 M acetic acid for 3 days and the resulting viscous solution was centrifuged at 12,000 × g for 1 h. The residue was re-extracted with 10 volumes of 0.5 M acetic acid for 3 days and the extracts were centrifuged at the same speed again. The supernatants were salted out by adding NaCl a final concentration of 0.7 M. After standing overnight, the resulting precipitate was collected by centrifuging at 12,000 × g for 1 h. The precipitate was dissolved in 10 volumes of 0.5 M acetic acid. Salting out and solubilization procedures were repeated 3 times. The resultant solution was dialyzed against 0.1 M acetic acid and lyophilized for further experiments. The lyophilized collagens were named as ASC, which is acid-soluble collagen. In addition, insoluble residues from pipefish were also lyophilized for preparing pepsin-solubilized collagen. The lyophilizates were suspended in 0.5 M acetic acid and digested with 1% (enzyme/substrates, w/w) pepsin (EC 3.4.23.1, 1:10,000, Sigma Chemical Co, USA) for 48 h. The pepsin-solubilized collagens were centrifuged at 12,000 × g for 1 h and the supernatants were dialyzed against 0.02 M Na2HPO4 (pH 7.2) for 3 days. After dialysis, the resultant was dissolved in 0.5 M acetic acid and salted out by adding

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Peptide mapping of collagen samples was performed according to the method of Kimura et al. [15] with a slight modification. Each collagen sample (0.2 mg) was dissolved in 0.1 M sodium phosphate buffer (pH 7.2) containing 0.5% SDS and heated at 100oC for 5 min. The digestion was carried out at 37oC for 30 min by adding 5 μL of lysyl endopeptidase from A. lyticus (EC 3.4.21.50; 4.5 amidase activity/mg protein; Wako, Osaka, Japan). After adding SDS to a final concentration of 2%, the proteolysis was stopped by boiling for 5 min. SDS-PAGE was performed by the method of Laemmli [14] using 12.5% gel. `jJÅÉääìäçëÉ=`çäìãå=`Üêçã~íçÖê~éÜó

To investigate each subunit composition of collagen sample, the samples were applied to a CM-cellulose column chromatography. Collagen samples were dissolved in 20 mM sodium acetate buffer (pH 4.8) containing 6 M urea at 4oC and then incubated at 45oC for 30 min. The solution was centrifuged at 20,000 × g for 1 h and the supernatant was applied to a CM-cellulose column (1.5 × 15 cm) previously equilibrated with the same buffer. Each subunit was eluted with a linear gradient of 0~0.1 M NaCl in the same buffer at a flow rate of 0.4 mL/min. The subunit component was detected by absorbance at 230 nm and the fractions indicated by the numbers were examined by SDS-PAGE. aÉíÉêãáå~íáçå=çÑ=aÉå~íìê~íáçå=qÉãéÉê~íìêÉ

The denaturation temperature (Td) was determined by the

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method of Kimura et al [15]. Five milliliters of 0.03% collagen solution in 0.1 M acetic acid was used for viscosity measurement. The flow rates used as an index for calculation of reduced viscosities were average of five observations. The relation is described as follows: relative viscosity (ηrel) = flow time of sample/flow time of control (0.1 M acetic acid); specific viscosity (ηsp) = ηrel - 1. The denaturation temperature was taken as the mid point of the linear portion of the sigmoidal curve obtained by plotting ηsp at toC against various temperatures. aÉíÉêãáå~íáçå=çÑ=^ãáåç=^ÅáÇ=`çãéçëáíáçå

Amino acids in isolated collagens were determined from a hydrolysate with 6 N HCl at 110oC for 24 h using amino acid auto-analyzer (Biochrom 20; Pharmacia Biotech Ltd, Cambridge, UK). Amino acids were determined by derivatization with ninhydrin and measurement of absorbance at 570 nm except for proline and hydroxyproline, for which absorbance at 440 nm was measured. The amino acid content was expressed by the number of residues per 1,000 residues. rsJsáë=péÉÅíê~

The UV-Vis adsorption spectra of collagens from bullfrog skin were recorded by a Cary 1-C UV-Visible spectrophotometer (Varian Inc., Australia). Data collection and plotting were accomplished by the UVPC program supplied by the manufacture.

RESULTS AND DISCUSSION fëçä~íáçå=çÑ=`çää~ÖÉå=Ñêçã=máéÉÑáëÜ

The seaweed pipefish collagen was hardly solubilized with the only treatment of 0.5 M acetic acid. The yield of acid-soluble collagen isolated from pipefish (ASC) was about 5.5%, on the basis of lyophilized dry weight. That value was similar to the results, 5.2% of collagen on the basis of dry weight was isolated from nautilus outer skin [16], and high to the results, 0.9% and 2% collagens were produced from minke whale [17] and cuttlefish skin [18], respectively. After the acid-treatment, the insoluble residue in 0.5 M acetic acid was subsequently treated with 10% (w/v) pepsin. The yield of pepsin-solubilized collagen from bullfrog muscle (PSC) was 33.2% (on the dry weight basis). This result was low to those for edible jellyfish exumbrella (46.4%) [19], rhizostomous jellyfish mesogloea (35.2%) [20], cuttlefish outer skin (35.0%) [21], and brown backed toadfish skin [22]. This result might suggest that there are some discrepancies in the construction of collagens among different species. For collagen molecule, the two terminal ends are non-helical parts, which play an important role in the cross-linked structure. If the molecules are highly crosslinked at the telopeptide region, the solubility of collagen will decrease [23]. It suggested the seaweed pipefish had abundant collagen and a large amount of collagen can be

cáÖK=NK SDS-Polyacrylamide gel electrophoresis patterns of collagens isolated from pipefish. Lane a, molecular weight markers; lane b, porcine skin collagen; lane c, ASC (acidsolubilized collagen from pipefish); and lane d, PSC (pepsinsolubilized collagen from pipefish).

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The two collagens (ASC and PSC) from pipefish were examined by SDS-PAGE, using a 6% gel (Fig. 1). SDS-PAGE pattern showed that all collagens were mainly composed of at least two different α chains, α1 and α2, and the density of α1 is higher than α2. The existence of a α3 chain was not identified under the electrophoretic conditions. Based on electrophoretic mobility and subunit composition, it was suggested that collagens from pipefish were similar to those of porcine skin collagen. Hence, a major component of collagens extracted from pipefish is type I collagens and were composed of two α1 and α2 chains. The results were in agreement with the previous reports [21,23]. mÉéíáÇÉ=j~ééáåÖ

To compare the patterns of peptide fragments with ASC (acid-solubilized collagen) and PSC (pepsin-solubilized collagen), the collagen hydrolysates digested by lysyl endopeptidase were migrated on 12.5% SDS-polyacrylamide gel (Fig. 2). The electrophoretic pattern of ASC (Fig. 2) was different pepsin solubilized collagen (PSC). At the same time, PSC could not be degraded, which meant the structure of PSC was much rigid against digestion by lysyl endopeptidase. pìÄìåáí=`çãéçëáíáçå=çÑ=máéÉÑáëÜ=`çää~ÖÉå

After chemical and thermal treatments with dissolution in 20 mM sodium acetate buffer, pH 4.8, containing 6 M urea at 4oC and incubation at 45oC for 30 min, the denatured ASC was applied to a carboxymethyl cellulose (CM-cellulose) column chromatography and it was resolved into two main

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^= = = = = = = = = = _= cáÖK=OK Peptide mapping of lysyl endopeptidase digests from collagens using 12.5% gel. (A) ASC (acid-solubilized collagen) and (B) PSC (pepsin-solubilized collagen from pipefish).

peaks corresponding to α subunits disentangled from the collagen fiber (Fig. 3A). To visualize α subunits, the several chromatographic fractions, as indicated by the fraction (A and B) were analyzed by SDS-PAGE. These results suggest that ASC consists of two α chains. Two protein fractions contained α chains as a major component; this collagen consisted of two α chains such as α1 (fraction A) and α2 (fraction B) in the order of their elution. As a result, the collagen (ACS) from pipefish had a chain composition of (α1)2α2 heterotrimer, similar to that of mammalian collagen, such as porcine skin. Nagai and Suzuki [21] reported that the acidsolubilized collagen from paper nautilus (Argonauta argo, Linnaeus) outer skin had a chain composition of (α1)2α2 heterotrimer. The PSC was also applied to CM-cellulose column chromatography and it was separated into three peaks, two having large peaks containing a α chain as a major component (Fig. 3B). This result suggests that this collagen consists of α chains. That is, these chains were α1 (fraction A), α2 (fraction B), and α3 (fraction B), respectively. As a result, the collagen (PCS) from pipefish had existence of two molecular forms of (α1)2α2 and α1α2α3 heterotrimer. Kimura (1988) reported that the α3 chain was widely distributed in the fish skin collagen. In addition, many researcher reports that a α3 chain was detected in 14 fish specis of 17 teleosts [24-26]. aÉå~íìê~íáçå=qÉãéÉê~íìêÉ

The denaturation temperature (Td) was determined by viscosity measurement. Relative viscosity of collagens in 0.1 M acetic acid is depicted in Fig. 4. The Td of ASC and PSC was about 34.8oC and 35.7oC, respectively. These results indicate that the Td of ASC was similar than that of PSC. In addition, the Td of PCS was similar than that of porcine skin collagen (37oC) [15], but higher than collagens originated from other

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cáÖK=PK CM-Cellulose column chromatography of denatured collagens. A, ASC and B, PSC. A 1.0 × 15 cm column of CMcellulose was equilibrated with 20 mM sodium acetate buffer (pH 4.8) containing 6 M urea and maintained at 4°C. The collagens (15 mg) was dissolved in 5 mL of the same buffer, denatured at 60°C for 15 min, and then eluted from the column with a linear gradient of 0~0.15 M NaCl at a flow rate of 0.4 mL/min. The fractions indicated were examined by SDSPAGE as shown in the inset.

marine organisms. For examples, the Td of collagen obtained from paper nautilus outer skin was 27.0oC [19] and collagens investigated in fish species such as sardine, red sea beam, Japanese sea bass, and cod skin were 28.5, 28.0, 28.0, and 31.5oC as the denaturation temperature (Td), respectively [27,17]. These results can be attributed to the physiological and biochemical nature of the pipefish. The heat transformation of collagen is interpreted as disintegration of the collagen triple helical structure into random coils. This is accompanied by a change in physical properties, such as viscosity, sedimentation, diffusion, light scattering, and optical activity [28].

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q~ÄäÉK=NK Amino acid composition of collagens isolated from pipefish (Residues/1,000 residues) Amino acid

cáÖK=QK Thermal denaturation curve of collagens isolated from pipefish. The denaturation temperature was measured by viscosity in 0.1 M acetic acid. The incubation time at each temperature was 30 min. Collagen concentration 0.03%. ASC, acidsoluble collagen; PSC, pepsin-solubilized collagen.

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Amino acid composition is expressed as residues per 1,000 total amino acid residues and is shown in Table 1. In amino acid profile, it is similar between the acid-soluble collagens (ASC) and pepsin-solubilized collagens (PSC). The two collagens contain glycine as the major amino acid, and that was about 31.5% and 30.3% of total amino acids, respectively. Because glycine represents approximately one third of the total residues of amino acids and could be found as every third residue in collagen molecules except for the first 10 amino acid residues from the N-terminus, and the first 10 amino acid residues from the C-terminus of the collagen molecules [22,29]. In addition, two collagens isolated from pipefish were relatively high contents of proline, hydroxylproline, alanine, glutamic acid, and aspartic acid, while methionine, valine, and threonine were low. Tryptophan and cystine were not detected. The higher the imino acid content, the more stable are the helices of collagen. The molecular structure of collagen is maintained mainly by restrictions on changes in the secondary structure of the polypeptide chain imposed by the pyrrolidine rings of proline and hydroxyproline, and also maintained partially by the hydrogen bonding ability of the hydroxyl group of hydroxyproline. The total contents of imino acid were 18.5% and 19.3% of ASC and PSC, similar to the collagens from fish scale (19.3~19.7%) [27], walleye pollock skin (18.4%) [30], and minke whale (19.9%) [17], but significantly higher than those of collagens from edible jelly fish exumbrella (12.2%) [18], ocellate puffer fishi skin (17.0%) [21], and channel catfish skin (17.0%) [31]. The dehydroxylation degrees of hydroxyproline in ASC and PSC were 44.3% and 45.1%, respectively. Hydroxylation of proline and lysine are important for the thermal sta-

Pipefish ASC

PSC

Aspartic acid

54

57

Threonine

15

16

Serine

36

36

Glutamic acid

73

80

Glycine

315

303

Alanine

90

91

Cystine

-

-

Valine

15

Methionine

8

14 5

Isoleucine

16

17

Leucine

32

30 25

Tyrosine

22

Phenylalanine

42

41

Histidine

28

26

Lysine

24

22 44

Arginine

45

Hydroxyproline

82

87

Proline

103

106

Imino acid

185

193

Total

1000

1000

bility of collagen since hydroxyproline stabilizes the triple helix of collagens [32]. In the previous papers, the degree of hydroxylation of proline in collagens measured from 32.8%~51.7% [4,18-21,30,31]. It appears that it was the lower imino acid content rather than the extent of hydroxylation that seems to be the reason for the lower denaturation temperature observed for collagens from equatic organisms. Fig. 5 shows UV-Vis spectra of collagens isolated from pipefish. It is generally known that tyrosine and phenylalanine are sensitive chromophores, which absorb light below 300 nm. Since tryptophan did not exit in the collagens from pipefish, but contents of tyrosine and phenylalanine were high, all collagens have the absorption peak at 280 nm. As a result, both ASC (Fig. 5A) and PSC (Fig. 5B) have adsorption near 230 nm and 280 nm, respectively, and the similar absorbance 229 nm and 276 nm of porcine skin collagens.

CONCLUSION Seaweed pipefish contains a large quantity of collagens so that pipefish have potential to be an important source of collagen without the threat of BSE and TSE. Collagen extracted mainly from skins and bones of cattle and pigs has been utilized in medicines, cosmetics, and food industry. Therefore, we isolated collagens of pipefish and characterized their various physicochemical properties such as subunit composition, denaturation temperature, UV-Vis spectra, and amino

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cáÖK=RK UV-Vis spectra of collagens isolated from pipefish. A, ASC and B, PSC.

acid contents to establish basic data for applications in industrial fields. ^ÅâåçïäÉÇÖÉãÉåíë= This research was supported by a grant (M2007-01) from Marine Bioprocess Research Center of the Marine Bio 21 Center funded by the Ministry of Maritime Affairs and Fisheries, Korea. This study was financially supported by Pukyong National University in the 2007 PostDoc. Program.

Received January 9, 2009; accepted March 6, 2009

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