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1 Korean J. Malacol. 27(1): 9-14, 2011 이매패류 3 종의당면체소화효소활성 주선미, 권오남 1, 김재원 2, 이정식 전남대학교수산생명의학과, 1 강릉원주대학교해양생물교육센터, 2 강원도립대학해양생명과학과 Digestive Enzyme Activity within Crystalline Style in Three Species of Bivalves Sun Mi Ju, O-Nam Kwon 1, Jae Won Kim 2 and Jung Sick Lee Department of Aqualife Medicine, Chonnam National University, Yeosu , Korea; 1 Marine Biology Center for Research and Education, Gangnung-Wonju National University, Gangneung , Korea; 2 Department of Marine Life-Science, Gangwon Provincial College, Gangneung , Korea ABSTRACT This study was examined digestive enzyme activity in the crystalline style of the three species of bivalves. Bivalves used in this study were Tegillarca granosa (n=61), Mytilus galloprovincialis (n=30) and Saxidomus purpuratus (n=30) and collected from southern coast of Korea on May Digestive enzymes activities in the crystalline style were assayed in spectrophotometer. Amylase and cellulase occupied approximately 90% of digestive enzyme in crystalline style of T. granosa, M. galloprovincialis, and S. purpuratus. And protease activity in crystalline style of T. granosa, M. galloprovincialis and S. purpuratus showed the lowest values to 0.02, 0 and 0.08%, respectively. Digestive enzyme activity in crystalline style of three species was measured in the order of cellulase > amylase > chitinase > laminarinase. Key Words: crystalline style, digestive enzymes, Tegillarca granosa, Mytilus galloprovincialis, Saxidomus purpuratus 서 이매패류의소화기작은주요먹이원인식물성부유생물들의세포벽을분해하기위한화학적소화가필요하다. 따라서이매패류들은당면체낭 (crystalline style sac) 에서다양한소화효소를함유한당면체 (crystalline style) 를만들어소화작용에이용한다 (Reid and Sweeney, 1980; Brock, 1989; Ibarrola et al., 1988; Alyakrinskaya, 2001). 이매패류의당면체를구성하는주요소화효소들은종에따라다소차이를보인다. Crassostrea virginica, Geukensia demissa, Macoma balthica, M. mitchlli, Rangia cuneata에서는 amylase, cellulase, laminarinase 등주로탄수화물분해효소들로구성된다 (Brocka and Kennedy, Received December 15, 2010; Revised December 20, 2010; Accepted January 12, 2011 Corresponding author: Sun Mi Ju Tel: +82 (61) mi357579@nate.com /24372 론 1992). 하지만 Cerastoderma edule (Ibarrola et al., 1998) 과 Ruditapaes decussatus 및 Venerupis pullastra 의당면체는탄수화물분해효소와 protease로구성된다 (Albentosa and Moyano, 2009). 당면체의소화효소학적기능에관한연구들은 Strombus gigas의간췌장과당면체에서적정온도와 ph에따른탄수화물분해효소의활성 (Horiuchi and Lane, 1966), Placopecten magellanicus의소화맹낭과당면체에서탄수화물분해효소의활성 (Wojtowicz, 1972), Telescopium telescopium의당면체구성과효소함유물 (Alexander et al., 1979), Mya arenaria 당면체의 amylase 활성 (Trainer and Tillinghast, 1982) 등이보고되었다. 하지만이와관련된국내연구로는복족류인 Nesiohelix samarangae의소화관에서 cellulase 활성에대한세포화학적및면역세포화학적연구 (Jeong et al., 1997, 1998) 가있을뿐이매패류당면체의소화효소에관한연구는전무하다. 따라서본연구에서는이매패류의화학적소화에초점을맞추어서식방법이서로다른꼬막, Tegillaca granosa, 지중해담치, Mytilus galloprovincialis 및개조개, Saxidomus - 9 -

2 이매패류 3 종의당면체소화효소활성 purpuratus를대상으로당면체의소화효소학적특징에대해조사하였다. 재료및방법 1. 재료분석에사용된이매패류는돌조개과 (Arcidae) 의꼬막, Tegillarca granosa, 홍합과 (Mytilidae) 의지중해담치, Mytilus galloprovincialis 및백합과 (Veneridae) 의개조개, Saxidomus purpuratus이다. 이들은 2010년 5월에남해안여수연안에서각각 61, 30, 30개체를채집하였다. 개체의크기와당면체의습중량은 Table 1에나타냈으며, 당면체의형태는 Fig. 1에나타냈다. 2. 방법 1) 효소활성분석당면체는증류수 (ice cooling, 0 ) 2 ml를넣고 1,800 rpm으로 1분간균질화하였다 (homogenizer, SHM-7211 YHaNa ). 이후 4, 6,000 rpm에서 30분동안원심분리하여상층액을조효소액으로이용하였다. 모든시료는각효소 분석용으로구분하여 -80 초저온냉동고에보관하였다. 당면체의소화효소활성분석은분광광도계 (Multiskan Thermo Scientific, U.S.A.) 를이용하여 amylase, laminarase, chitinase, cellulase는최대흡수파장인 540 nm에서흡광도를측정하였고, protease는 410 nm에서흡광도를측정하였다. Amylase 활성은 Bernfeld (1955) 의방법을이용하여분석하였으며, laminarase 활성은 Somogyi (1952) 의방법, chitinase 활성은 Monreal and Reese (1969) 의방법을이용하여분석하였다. 그리고 cellulase 활성은 DNS procedure (Miller et al., 1960) 를따랐으며, Trypsin 활성은 Erlanger method (Erlanger et al., 1961) 의방법을이용하였고, TG-Lipase 활성은 p-npp method (Hung et al., 2003) 의방법을이용하여분석하였다. 사용된기질, 반응온도, 시간그리고완충용액의농도와종류는 Table 2에나타냈다. 단백질은 BSA를표준물질로사용하는 Bradford and Reid (1967) 방법을이용하여정량하였다. 2) 통계분석 Table 1. Size of specimen and weight of crystalline style used in analysis Species Tegillarca granosa Mytilus galloprovincialis Saxidomus purpuratus Shell size (mm) Total weight (g) Body weight (g) Cs* weight (mg, wet weight) SL ± ± ± ± SH ± ± ± ± SL ± ± ± ± *Cs, crystalline style; SH, shell height; SL, shell length. Fig. 1. Morphology of crystalline style of Tegillarca granosa (A), Mytilus galloprovincialis (B) and Saxidomus purpuratus (C). Aam, anterior adductor muscle; Am, adductor muscle; Cs, crystalline style; Dg, digestive gland; Es, exhalent siphon; F, foot; Fi, filament; Is, inhalent siphon; M, mantle; Pam, posterior adductor muscle; Sa, stomach; Ss, crystalline style sac. Scale bar: 5 mm

3 Korean J. Malacol. 27(1): 9-14, 2011 Table 2. Absorptiometry of digestive enzyme analysis of crystalline style Enzyme ph Buffer Temp. ( ) Incubation time (min) Standard Supernatant volume (μl) Protein assay Amylase M citrate phosphate Maltose (5-200 μg/ml) 50 4 Laminarinase 6.0 Chitinase 6.0 Cellulase M sodium acetate buffer 0.2M potassium phosphate buffer 50mM sodium citrate Glucose (0.1%) NAG* Glucose 50 4 Bradford and Reid (1967) Protease M potassium phosphate buffer Tyrosine ( μg/ml) 50 4 * 0.1% NAG, N-Acetyl-D-Glucosamine 분석자료의유의성은 One-way ANOVA test를실시하여 Duncan's multiple range test (Duncan, 1955) 로처리한후평균간의유의성 (P < 0.05) 을 SPSS program (Ver., 14.0) 으로검정하였다. 났으며 (Fig. 3), 개조개에서는 cellulase (63.08%) > amylase (28.91%) > chitinase (7.88%) > laminarase (0.05%) 의순으로나타났다 (Fig. 4). 결 과 꼬막, 지중해담치, 개조개의당면체를구성하는소화효소는 amylase와 cellulase가약 90% 로대부분을차지하였다. 그리고꼬막, 지중해담치, 개조개의당면체를구성하는소화효소중 protease의활성도가가장낮았으며, 각각 0.02, 0, 0.08% 로나타났다 (Figs. 2, 3, 4). 꼬막의당면체를구성하는소화효소활성도는 cellulase (67.55%) > amylase (20.56%) > chitinase (11.75%) > laminarase (0.12%) 의순으로나타났다 (Fig. 2). 지중해담치에서는 cellulase (57.82%) > amylase (31.54%) > chitinase (10.51%) > laminarase (0.13%) 의순으로나타 Fig. 3. Enzymatic activity of crystalline style in Mytilus galloprovincialis. Vertical bar: SD. Fig. 2. Enzymatic activity of crystalline style in Tegillarca granosa. Vertical bar: SD. Fig. 4. Enzymatic activity of crystalline style in Saxidomus purpuratus. Vertical bar: SD

4 이매패류 3 종의당면체소화효소활성 고찰이매패류의섭이및소화기작은외투강기관계와내장낭기관계의작용에의해수행된다. 이매패류의내장낭내에서의소화기작은물리적소화보다는이들의주요먹이원인식물성부유생물들의세포벽을분해하기위한화학적소화기작이필요하다. 따라서이매패류들은당면체낭에서셀룰로스분해효소를함유한당면체를만들어소화작용에이용하게된다 (Alyakrinskaya, 2001). 본연구에서는이매패류의소화기작가운데화학적소화에초점을맞추어서식방법이서로다른꼬막, Tegillarca granosa, 지중해담치, Mytilus galloprovincialis 및개조개, Saxidomus purpuratus를대상으로화학적소화와직접적인관련이있는당면체의소화효소의활성을조사하였다. 3종모두당면체를구성하는소화효소는 amylase와 cellulase가약 90% 내외를차지하였으며, chitinase는 %, laminarinase는 % 를차지하였다. 고착성인 Crassostrea virginica, Rangia cuneata 그리고 Geukensia demissa와잠입성인 Macoma balthica와 M. mitchelli의당면체에서 amylase의활성이전체탄수화물분해효소의활성중 30-70% 로높은비율을나타냈고, cellulase 는 15-27%, laminarinase는 14-47% 를나타냈다. 그리고 3 가지탄수화물분해효소의활성은서식지가다른종간에유사하였으며, 소화맹낭에서보다당면체에서활성이더높았다. 이는당면체의소화효소는이매패류의세포외소화에서중요한역할을수행하는것뿐만아니라세포내소화에도관여하기때문이라고하였다. 즉세포내탄수화물분해효소활성은당면체에의해분비된효소와관련이있다는것이다 (Brocka and Kennedy, 1992). Fernández-Reiriz et al. (2001) 은 Mytilus chilensis를인위적으로서식지를옮겨서식지적응상태를알아보는실험에서분석한소화효소의활성중 cellulase가가장높게나타났다. 또한 Albentosa and Moyano (2009) 의연구결과, 조하대에서식하는백합과이매패류인 Venerupis pullastra 의당면체에서 amylase, cellulase, laminarase 중 amylase 활성이 75.4% 로가장높았고조간대에서식하는 Ruditapaes decussatus의경우 amylase 활성이 92.6% 에달했다. 또한 protease/amylase ratio는 Ruditapaes decussatus보다 V. pullastra에서매우높았다. 이는식물성부유생물의양이조하대보다조간대에많이있기때문이라고하였다. Albentosa and Moyano (2009) 는 amylase 활성이 cellulase 활성보다현저히높은반면 Fernández-Reiriz et al. (2001) 은 cellulase 활성이 amylase 활성보다 2배가량높은활성을보일뿐만아니라전체탄수화물분해효소중 % 를 차지하였다. 위의두연구결과에서이러한효소활성의차이는먹이조성의차이로생각된다. 이매패류에서 chitinase의활성에대한연구는 Crassostrea virginica의당면체에서 chitinase의활성 (Smucker and Wright, 1984), Pecten maximus (Stark and Walker, 1983) 와 Placopecten magellanicus (Wojtowicz, 1972) 의소화선에서 chitinase의활성을제외하고는거의찾아보기가힘들다. 두가리비의소화선에서 chitinase는검출되지않았다. 또한 Sumner (1969) 는 Mytilus edulis 소화세관의소화세포의세포질과립들에서강한 chitobiase 활성을발견하였는데이는 1차라이소좀에의한것이라고하였다. 그리고 Owen (1966) 은연체동물에서키틴가수분해는소화관에존재하는세균에의해일어난다고하였다. 본연구결과, 3종이매패류의당면체에서 laminarinase는 % 로매우낮았다. 하지만 Placopecten magellanicus의당면체와소화맹낭에서 laminarinase는높은활성을보였다 (Wojtowicz, 1972). 꼬막, 지중해담치, 개조개의당면체를구성하는효소중 protease의활성도는각각 0.02, 0, 0.08% 로탄수화물분해효소와비교해서낮게나타났다. 당면체가아미노산과탄수화물성분으로구성되었다는사실이알려졌다 (Bailey and Worboys, 1960; Doyle, 1966; Albentosa and Moyano, 2009). 그러나여러저자들이당면체의효소활성에대한분석을진행하기는했지만 (Horiuchi and Lane, 1966; Wojtowicz, 1972; Trainer and Tillinghast, 1982), Judd (1987) 를제외하고는당면체의단백질에대한연구는거의없다. Judd (1987) 는당면체는당단백질로구성되어있으며, 단단하고탄력이있는당면체를갖는 Macomona liliana, Pholadidea spathulata 그리고 Perna canaliculus와부드럽고탄력이없는당면체를갖는 Crassostrea glomerata, Chione stutchburyi 두그룹간의종들에서단백질양상사이에차이점은없다고보고하였다. Reid (1966) 는 Lima hians에관한연구에서지방과단백질소화는주로소화맹낭의소화세관의소화세포에서세포내소화가일어나는것을발견하였고, 반면에 Mya arenaria 에서단백질과지방가수분해는소화세관의소화세포뿐만아니라위에서세포외소화가일어나는것을발견하였다. 이렇게당면체에저장된소화효소들의활성이서로다른이유는서식지뿐만아니라먹이생물의종류및구성성분에따라변하기때문이다 (Seiderer et al., 1982; Fernández-Reiriz et al., 2001; Albentosa and Moyano, 2009). 본연구에사용된이매패류 3종의서식환경을보면꼬막은조간대부터수심 10 m까지의진흙, 개조개는조하대수심

5 Korean J. Malacol. 27(1): 9-14, 2011 m 의모래가섞인진흙그리고지중해담치는조간대부터수심 20 m 사이의기질에부착하여서식한다 (Yoo, 1976). 이들의각각영양조성을 Yoon (2003), Noh et al. (2006), Fernández-Reiriz et al. (2001) 을기준으로유추해보면, 전체적으로탄수화물분해효소활성과단백질분해효소활성이차이를보이는것은탄수화물을주에너지원으로이용하는이매패류의서식지및먹이생물의종류와연관이있음을의미한다. 하지만, 이들의소화효소의활성을더면밀히조사하기위해서는소화맹낭과당면체의소화효소의활성비교와 SDS-PAGE에의한효소종류별 isomer에대한연구가필요할것으로판단된다. 요 본연구는 3종의이매패류를대상으로당면체의소화효소활성에대해조사하였다. 본연구에사용된이매패류는꼬막 (n=61), 지중해담치 (n=30) 및개조개 (n=30) 이며, 이들은한국남해안에서 2010년 5월에채집하였다. 당면체의소화효소활성분석은분광광도계를이용하였다. 꼬막, 지중해담치, 개조개의당면체를구성하는소화효소는 amylase와 cellulase가약 90% 로대부분을차지하였다. 그리고꼬막, 지중해담치, 개조개의당면체를구성하는소화효소중 protease 의활성도가가장낮았으며, 각각 0.02, 0, 0.08% 로나타났다. 당면체를구성하는소화효소활성도는 3종모두 cellulase > amylase > chitinase > laminarinase의순으로나타났다. 약 감사의글 본연구는지역미래과학기술여성인력양성사업 ( 조선대학교 WISE 광주 / 전남지역센터 ) 과지식경제부지방기술혁신사업 (RTI ) 지원으로수행되었으며, 이에감사드립니다. REFERENCES Alexander, C.G., Cutler, R.L. and Yellowless, D. (1979) Studies on the composition and enzyme content of the crystalline style of Telescopium telescopium (L.) (Gastropoda). Comp. Biochem. Physiol., Part B, 64(1): Albentosa, M. and Moyano, F.J. (2009) Differences in the digestive biochemistry between the intertidal clam, Ruditapes decussatus, and the subtidal clam, Venerupis pullastra. Aquacult. Int., 17: Alyakrinskaya, I.O. (2001) The dimensions, characteristics and functions of the crystalline style of Molluscs. Biol. Bull., 28(5): Bailey, K. and Worboys, B.D. (1960) The lamellibranch crystalline style. Biochem. J., 76: Bernfeld, P. (1955) Amylases, alpha and beta. Meth. Enzymol., 1: Bradford, M.M. (1967) A rapid and sensitive method for the quantitation of microgram quantities of characterization of a strong fibrinolytic enzyme (nattokinase) in the vegetable cheese bovine insulin. Biochemistry, 6: Brock, V. (1989) Crassostrea gigas (Thunberg) hepatopancreas-cellulase kinetics and cellulolysis of living monocellular algae with cellulose walls. J. Exp. Mar. Biol. Ecol., 128(2): Brocka, V. and Kennedy, V.S. (1992) Quantitative analysis of crystalline style carbohydrases in five suspension- and deposit-feeding bivalves. J. Exp. Mar. Biol. Ecol., 159(1): Doyle, J. (1966) Studies on the chemical nature of the crystalline style. In: Some contemporary studies in marine science (ed. by Barnes, H.), George Allen and Unwin, London, pp Erlanger, B.F., Kokowski, N. and Cohen, W. (1961) The preparation and properties of two new chromogenic substrates of trypsin. Arch. Biochem. Biophy., 95: Fernández-Reiriz, M.J., Labarta, U., Navarro, J.M. and Velasco, A. (2001) Enzymatic digestive activity in Mytilus chilensis (Hupé 1854) in response to food regimes and past feeding history. J. Comp. Physiol., 171: Horiuchi, S. and Lane, C.E. (1966) Carbohydrases of the crystalline style and hepatopancreas of Strombus gigas linné. Comp. Biochem. Physiol., Part A, 17(4): Hung, T.C., Giridgar, R., Chiou, S.G. and Wu, W.T. (2003) Binary immobilization of Candida rugosa lipase on chitosan. J. Mol., Catal., B Enzym., 26: Ibarrola, I., Larretxea, X., Iglesias, J.I.P., Urrutia, M.B. and Navarro, E. (1998) Seasonal variation of digestive enzyme activities in the digestive gland and the crystalline style of the common cockle Cerastoderma edule. Comp. Biochem. Physiol., Part A, 121: Jeong, K.H. and Lee, Y.S. (1997) Cytochemical and immunocytochemical study on the cellulase activity in the stomach of the land snail Nesiohelix samarangae. Korean J. Malacol., 13(2): Jeong, K.H., Lee, Y.S. and Kim, E.J. (1998) Cytochemical and immunocytochemical study on the cellulase activity in the digestive tract of the land snail Nesiohelix samarangae. Korean J. Malacol., 14(2): Judd, W. (1987) Crystalline style proteins from bivalve molluscs. Comp. Biochem. Physiol., Part B, 88(1): Miller, G.G., Blum, R., Glennon, W.E. and Burton, A.L. (1960) Measurement of carboxymethylcellulase activity. Anal. Biochem., 1: Monreal, J. and Reese, E.T. (1969) The chitinase of Serratia marcescens. Can. J. Microbiol., 15:

6 이매패류 3 종의당면체소화효소활성 Noh, I.H., Yoon, Y.H, Park, J.S., Soh, H.Y. and Kim, D.-I. (2006) Spatio-temporal distributions of organic matter in surface sediment in the central part of the South sea, Korea. J. Korean Soc. Mar. Environ. Engin., 9: Owen, G. (1966) Digestion. In: Physilogy of Mollusca (ed. by Wilbur, K.M. and Yonge, C.M.) Vol. II, Academic Press, New York, pp Reid, R.G.B. (1966) Digestive tract enzymes in the bivalves Lima hians (Gmelin) and Mya arenaria L. Comp. Biochem. Physiol., 17: Reid, R.G.B. and Sweeney, B. (1980) The digestibility of the bivalve crystalline style. Comp. Biochem. Physiol., Part B, 65(2): Somogyi, M. (1952) Notes on sugar determination. J. Biol. Chem., 195: Seiderer, L.J., Newell, R.C. and Cook, P.A. (1982) Quantitative significance of style enzymes from two marine mussels (Choromytilus meridiomalis Krauss and Perna perna Linnaeus) in relation to diet. Mar. Biol. Lett., 3: Smucker, R.A. and Wright, D.A. (1984) Chitinase activity in the crystalline style of the American oyster Crassostrea virginica. Comp. Biochem. Physiol., Part A, 77(2): Stark, J.R. and Walker, R.S. (1983) Carbohydrate digestion in Pecten maximus. Comp. Biochem. Physiol., Part B, 76(1): Sumner, A.T. (1969) The distribution of some hydrolytic enzymes in the cells of the digestive gland of certain lamellibranchs and gastropods. J. Zool., Lond., 158: Trainer, D.G. and Tillinghast, E.K. (1982) Amylolytic activity of the crystalline style of Mya arenaria (Bivalvia, Mollusca). Comp. Biochem. Physiol., Part A, 72(1): Wojtowicz, M.B. (1972) Carbohydrases of the digestive gland and the crystalline style of the Atlantic deep-sea scallop (Placopecten magellanicus Gmelin). Comp. Biochem. Physiol., Part A, 43(1): Yoo, J.S. (1976) Korean shells in color. Iljisa Publishing Co., Seoul, pp Yoon, Y.H. (2003) Spatio-temporal distribution of organic matters in surface sediments and its origin in Deukryang bay, Korea. J. Environ. Sci., 12:

19(4)-12(40).fm

19(4)-12(40).fm J. of Aquaculture Vol. 19(4) : 288-298, 2006 µ Journal of Aquaculture Korean Aquaculture Society s ½, «1, x 2 * w ûw, 1 w š w, 2 w w Distribution of Bivalve Larvae in Deukryang Bay Chul Won Kim, Seung