Aquaculture Science , Preparation of Abalone Enzymes Capable of Cell Wall Degradation of a Marine Red Alga Porphyra yezoens

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1 アワビ消化管由来の海産紅藻スサビノリ細胞壁分解酵素祖の調製誌名水産増殖 = The aquiculture ISSN 著者巻 / 号大塚, 周二福田, 覚柴田, 大輔ほか2 名, 54 巻 1 号掲載ページ p 発行年月 2006 年 3 月農林水産省農林水産技術会議事務局筑波産学連携支援センター Tsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research Council Secretariat

2 Aquaculture Science , Preparation of Abalone Enzymes Capable of Cell Wall Degradation of a Marine Red Alga Porphyra yezoensis (Bangiales, Rhodophyta) Shuuji OOTSUKA 1, Satoru FUKUDA 1, 2, Daisuke SHIBATA 1, Naotsune SAGA 1 and Takao OJIMA 1 Abstract: We extracted a xylanase and a mannanase from Ezo abalone Haliotis discus hannai, and examined the enzyme activity and the ability for cell wall degradation of Porphyra yezoensis. Abalone xylanase was partially purified from the crude enzyme prepared from the digestive fluid by column chromatography on a TOYOPEARL CM-650M. Abalone mannanase was successfully purified from the crude enzyme by column chromatographies on TOYOPEARL CM-650M, hydroxyapatite, and TOYOPEARL HW-50F. The gametophytic thalli of P. yezoensis could not be degraded by the xylanase alone. The purified mannanase could degrade the thalli into cell masses consisting of cells; however, it produced practically no protoplasts from the thalli. When the thalli were incubated both with the xylanase and the purified mannanase, some protoplasts could be produced from the thalli. The two enzymes may be useful for the studies on cell engineering and asexual seedling production of this alga. Key words: Haliotis discus hannai; Porphyra yezoensis; Mannanase; Xylanase Porphyra yezoensis Ueda Rhodophyta Rhodophyceae Protoflorideophycidae Bangiales Bangiaceae Porphyra 2005 Noda et al Ren et al Graduate School of Fisheries Sciences, Hokkaido University, Minato 3-1-1, Hakodate, Hokkaido , Japan 2 Kyowa Algatech Kyowa, Kyowa Concrete Industry Co., Ltd., Benten 24-13, Hakodate, Hokkaido , Japan 1991; 2001 Saga et al ,4- -1,3-3 -1,4- -1,3-1997

3 1991; Haliotis discus hannai 6 8 cm 10 1 ml 500 ml 10 mm ph ,000 g 30 Locust bean gum from Ceratonia siliqua seeds SIGMA 5 mg/ml Xylan from Oat Spelts Wako 20 mg/ml 10 mm ph ml 0.5 l ml ml 0.1 SDS Park-Johnson Park and Johnson unit1 1 mol 25 ml 10 mm ph 7.0 TOYOPEARL CM-650M 25 mm 265 mm M NaCl 1 5 ml TOYOPEARL CM-650M Fraction mm ph mm 200 mm M ph ml Fraction Apollo Centrifugal Concentrator 20 ml Orbital Biosciences 3 ml TOYOPEARL HW-50F 20 mm 900 mm 2 mg/ml CMC Carboxymethyl cellulose sodium salt, medium viscosity ICN Biomedicals Glucomannan from Konjac Tuber Wako -1,4-1,4- -D-Mannan, Megazyme 5 mg/ml 10 mm ph ml 0.5 ml ml ml 0.1 SDS Park-Johnson Park and Johnson unit1 1 mol TU-1 Kuwano et al E/m 2 /s 14ESL Kitade et al.

4 Absorbance at 280 nm ( ) Enzyme Activity () 0.15 NaCl (M) (---) Fraction Number (5.0 ml/tube) 0 0 Fig. 1. TOYOPEARL CM-650M column chromatography of abalone crude enzyme., absorbance at 280 nm;, mannanase activity;, xylanase activity. A, fraction A consisting of fraction 59-63; B, fraction B consisting of fraction ,000 ml TU-2, 0.2 m 250 ml/min 7 15 mm 1 ml 5 unit 0.7 M 22 4 Sealife Calcofluor White M2R, SIGMA Maeda and Ishida ml TOYOPEARL CM-650M Fig. 1 Fraction A Fig. 1 Fraction B A TOYOPEARL HW-50F Fraction SDS-PAGE 39, U/mg ,4- CMC -1,4- Fig. 2 Fig. 2. Substrate specificity of the purified abalone mannanase. Abalone mannanase activity was assayed with locust bean gum ( ), -1,4-mannan ( ), glucomannan ( ), agarose ( ), xylan ( ) and CMC ( ).

$yezoensis degraded by the fraction A$ including mannanase and xylanase.

5 (A) (B) (C) (D) (E) (F) (G) Fig. 3. Degradation of thalli from Porphyra yezoensis with abalone enzymes. A, thalli of P. yezoensis. B and C, protoplasts from thalli of P. yezoensis degraded by abalone crude enzyme. D and E, thalli of P. yezoensis degraded by the fraction A including mannanase and xylanase. F and G, thalli of P. yezoensis degraded by the purified mannanase. C, E and G, stained with Calcoflour white. A B 15 mm Fig. 3B, C A 4 Fig. 3D, E B Fig. 3F, G A

6 10 20 Bangia atropurpurea Araki et al A B -1,4- -1, A DNA DNA 2003 n2n n Kitade et al Mizuta et al B Haliotis discus hannai Porphyra yezoensis TOYOPEARL CM-650M TOYOPEARL CM-650M TOYOPEARL HW-50F COE 1997 pp

7 Araki, T., M. Hayakawa, Y. Tamaru, K. Yoshimatsu and T. Morishita (1994) Isolation and regeneration of haploid protoplasts from Bangia atropurpurea (Rhodophyta) with marine bacterial enzymes. J. Phycol., 30, pp DNA Kitade, Y., S. Fukuda, M. Nakajima, T. Watanabe and N. Saga (2002) Isolation of a cdna encoding a homologue of actin from Porphyra yezoensis (Rhodophyta). J. Appl. Phycol., 14, Kitade, Y., S. Fukuda and N. Saga (1999) Preliminary study on early development of monospore of Porphyra yezoensis (Bangiales, Rhodophyta). Fish Genet. Breed. Sci., 28, Kuwano, K., Y. Aruga and N. Saga (1996) Cryopreservation of clonal gametophytic thalli of Porphyra (Rhodophyta). Plant Sci., 116, Maeda, H. and N. Ishida (1967) Specificity of binding of hexopyranosyl polysaccharides with a fluorescent brightener. J. Biochem., 62, pp Mizuta, H., H. Yasui and N. Saga (2003) A simple method to mass produce monospores in the thallus of Porphyra yezoensis Ueda. J. Appl. Phycol., 15, pp Noda, H., H. Amano, K. Arashima, S. Hashimoto and K. Nishizawa (1989) Antitumour activity of polysaccharides and lipids from marine algae. Nippon Suisan Gakkaishi, 55, Park, J. T. and M. J. Johnson (1949) A submicrodetermination of glucose. J. Biol. Chem., 181, Ren, D., H. Noda, H. Amano, T. Nishino and K. Nishizawa (1994) Study on antihypertensive and antihyperlipidemic effects of marine algae. Fish. Sci., 60, Saga, N., M. Polne-Fuller and A. Gibor (1986) Protoplasts from seaweeds: production and fusion. Beihefte Zur Nur Nova Hedwigia, 83, pp

農林水産業における自然エネルギーの効率的利用技術に関する総合研究（グリーンエナジー計画）第1期成果の概要

( グリーンエナジー計画 ) 第一期成果の概要誌名農林水産業における自然エネルギーの効率的利用技術に関する総合研究著者農林水産技術会議事務局, 掲載ページ p. 1-207 発行年月 1982 年 3 月農林水産省農林水産技術会議事務局筑波事務所 Tsukuba Office, Agriculture, Forestry and Fisheries Research Council Secretariat