**12. References**


[3] Woodward, R.B. (1964) The structure of tetrodotoxin. *Pure Appl. Chem.* 9:49-74.

<sup>\*</sup> Corresponding Author

	- [4] Goto, T., Kishi, Y., Takahashi, S., Hirata, Y. (1965) Tetrodotoxin. *Tetrahedron* 21:2059-2088.

Instrumental Analysis of Tetrodotoxin 269

[23] Asakawa, M., Toyoshima, T., Shida, Y., Noguchi, T., Miyazawa, K.(2000) Paralytic toxins in a ribbon worm *Cephalothrix* species (Nemertean) adherent to cultured oysters

[24] Yasumoto, T., Nakamura, M., Oshima, Y., Takahata, J. (1982) Construction of a

[25] Nakamura, M., Yasumoto, T. (1985) Tetrodotoxin derivatives in puffer fish. *Toxicon*

[26] Yasumoto, T., Yotsu, M., Murata, M., Naoki, H. (1988) New tetrodotoxin analogues

[27] Khora, S. S., Yasumoto, T. (1989) Isolation of 11-oxotetrodotoxin from the puffer

[28] Endo, A., Khora, S.S., Murata, M., Naoki, H., Yasumoto, T. (1988) Isolation of 11 nortetrodotoxin-6(R)-ol and other tetrodotoxin derivatives from the puffer *Fugu* 

[29] Yotsu, M., Hayashi,Y., Khora, S.S., Sato, S., Yasumoto, T. (1992) Isolation and structural assignment of 11-nortetrodotoxin-6(S)-ol from the puffer *Arothron nigropunctatus*. *Biosci.* 

[30] Kotaki, Y., Shimizu, Y. (1993) 1-hydoxy-5, 11-dideoxytetrodotoxin, the first N-hydroxy and ring-deoxy derivative of tetrodotoxin found in the newt *Taricha granulosa*.

[31] Yotsu-Yamashita, M., Yamagishi, Y. Yasumoto, T. (1995) 5, 6, 11-trideoxytetrodotoxin

[32] Yotsu-Yamashita, M., Schimmele, B., Yasumoto, T. (1999) Isolation and structural assignment of 5-deoxytetrodotoxin from the puffer fish *Fugu poecilonotus*. *Biosci. Biotech.* 

[33] Yotsu, M., Endo, A., Yasumoto, T. (1989) An improved tetrodotoxin analyzer. *Agric.* 

[34] Yotsu-Yamashita, M., Meds, D., Kwet, A., Schneider, M. (2007). Tetrodotoxin and its analog 6-*epi*tetrodotoxin in newts (*Triturus* spp.; Urodela, Salamandridae) from

[35] Nagashima, Y., Maruyama, J., Noguchi, T., Hashimoto, K. (1987) Analysis of paralytic shellfish poison and tetrodotoxin by ion-pairing high performance liquid

[36] Asakawa,M., Delan,G.G., Tsuruda,S., Shimomura,M., Shida,Y., Taniyama,S., Quilantang, M.B., Shindo,J. (2010) Toxicity assessment of the xanthid crab *Demania*

[37] Noguchi, T., Jeon, J.K., Arakawa,O., Sugita,H., Deguchi,Y., Shida, Y., Hashimoto, K. (1986) Occurrence of tetrodotoxin and anhydrotetrodotoxin in *Vibrio* sp. isolated from

[38] Narita, H., Noguchi, T., Maruyama,J., Nara, M., Hashimoto, K. (1984) Occurrence of a tetrodotoxin-associated substances in a gastropod, "Hanamushirogai" *Zeuxis* 

*cultripes* from Cebu Island, Philippines. *J.Toxicology*., Article ID172367.

the intestines of xanthid crab, *Atergatis floridus*. *J.Biochemistry*, 99:311-314.

from the puffer fish *Fugu poecilonotus*. *Tetrahedron Lett.* 36: 9329-9332.

in HiroshimaBay, Hiroshima Prefecture, Japan. *Toxicon* 38, 763–773.

continuous tetrodotoxin analyzer. *Bull.Jpn.Soc.Sci.Fish.*, 48:1481-1483.

from the newt *Cynops ensicauda*. *J.Am.Chem. Soc.*, 110:2344-2345.

*Arothron nigropunctatus*. *Tetrahedron Lett.* 30: 4393-4394.

*niphobles*. *Tetrahedron Lett.* 29: 4127-4128.

*Biotech. Biochem.* 56: 370-371.

*J.Am.Chem.Soc.* 115: 827-830.

*Biochem.* 63: 961-963.

*Biol. Chem.*, 53: 893-895.

southern Germany. *Toxicon* 50: 306-309.

*siquijorensis*. *Bull.Jpn.Soc.Sci.Fish,* 50:85-88.

chromatography. *Nippon Suisan Gakkaishi*, 53:819-823.

23:271-276.


[23] Asakawa, M., Toyoshima, T., Shida, Y., Noguchi, T., Miyazawa, K.(2000) Paralytic toxins in a ribbon worm *Cephalothrix* species (Nemertean) adherent to cultured oysters in HiroshimaBay, Hiroshima Prefecture, Japan. *Toxicon* 38, 763–773.

268 Chromatography – The Most Versatile Method of Chemical Analysis

Scientific Societies Press, Tokyo, pp.70-91.

*Toxicol.-Toxin Rev*. 20: 1-10.

*Suisan Gakkaishi*, 57:163-167.

*J.Food Hyg. Soc.*, 40:391-395.

*Jpn. Soc.Sci.Fish.*, *37:642*-647.

tetrodotoxin. *Science* 199:188-189.

*Japan. Soc. Sci. Fish*., 47:935-941.

Association, Tokyo, pp. 232–239.

[4] Goto, T., Kishi, Y., Takahashi, S., Hirata, Y. (1965) Tetrodotoxin. *Tetrahedron* 21:2059-2088.

[6] Noguchi, T., Ebesu, J.S.M. (2001) Puffer poisoning: epidemiology and treatment. *J.* 

[7] Hashimoto, Y. (1979) Marine Toxins and Other Bioactive Marine Metabolites. Japan

[8] Khora, S.S., Isa, J., Yasumoto, T. (1991) Toxicity of puffers from Okinawa, Japan, *Nippon* 

[9] Mahmud, Y., Tanu, M.B, Takatani, T., Asayama, E., Arakawa. O., Noguchi, T. (2001) *Chelonodon patoca*, a highly toxic marine puffer in Japan. *J. Nat Toxins*, 10:69-74. [10] Mahmud, Y., Yamamori, K., Noguchi, T. (1999) Occurrence of TTX in a brackishwater puffer "Midorifugu" *Tetraodon nigroviridis*, collected from Thailand. *J.Food Hyg. Soc.*, 40:363-367. [11] Mahmud, Y., Yamamori, K., Noguchi, T. (1999) Toxicity and tetrodotoxin as the toxic principle of a brackishwater puffer *Tetraodon steindachneri*, collected from Thailand,

[12] Hwang, D.F, Kao, C.Y., Yang, H.C., Jeng, S.S., Noguchi, T., Hashimoto, K. (1992)

[13] Mahmud, Y., Tanu, M.B., Noguchi, T. (1999) First occurrence of a food poisoning incident due to ingestion of *Takifugu oblongus*, along with a toxicological report on three

[14] Mosher,H.S., Fuhrman,F.A., Buchwald,H.G., Fischer, H.G. (1964) Tarichatoxin -

[15] Noguchi, T., Kao, H., Hashimoto, Y. (1971) Toxicity of the goby, *Gobius criniger*. *Bull.* 

[16] Kim, Y.H., Brown,G.B., Mosher, H.S.,Furman, F.A. (1975) Tetrodotoxin : occurrence in

[17] Sheumack, D.D., Howden, M.E.H., Spence, I., Quinn, R.J. (1978) Maculotoxin: a neurotoxin from the venom glands of the octopus *Hapalochlaena maculosa* identified as

[18] Narita,H., Noguchi, T., Maruyama,J, Ueda,Y., Hashimoto, K., Watanabe, Y., Hida,K. (1981) Occurrence of tetrodotoxin in a trumpet shell "boshubora" , *Charonia sauliae*. *Bull.* 

[19] Noguchi,T., Maruyama,J., Ueda,Y., Hashimoto,K. Harada,T. (1981) Occurrence of tetrodotoxin in the Japanese ivory shell *Babylonia japonica*. *Bull. Jpn. Soc.Sci.Fish.*, 47:909-913. [20] Noguchi, T., Maruyama, J., Narita, H., Hashimoto, K. (1984) Occurrence of tetrodotoxin

[21] Noguchi, T. Narita, H. Maruyama, J. Hashimoto, K. (1982) Tetrodotoxin in the starfish *Astropecten polyacanthus*, in association with toxification of a trumpet shell,

[22] Kawabata, T., 1978. In: Ministry of Health and Welfare of Japan, (Ed.), Assay Method for Tetrodotoxin, Food Hygiene Examination Manual, vol. 2. Japan Food Hygiene

in the gastropod mollusk *Tutufa lissostoma* (frog shell). *Toxicon* 22:219-226.

"boshubora", *Charonia sauliae*. *Bull. Japan. Soc. Sci. Fish*. 48, 1173-1177.

Toxicity of puffer in Taiwan, *Nippon Suisan Gakkaishi*, 58:1541-1547.

marine puffer species in Bangladesh. *J.Food Hyg. Soc.*, 40:473-480.

tetrodotoxin, a potent neurotoxin. *Science* 144: 1100-1110.

Atelopid frogs of Costa Rica. *Science* 189:151-152.

[5] Narahashi, T. (2001) Pharmacology of tetrodotoxin. *J. Toxicol.-Toxin Rev.* 20:67-84

	- [39] Asakawa, M., Toyoshima,T., Ito, K., Bessho, K., Yamaguchi, C., Tsunetsugu, S., Shida, Y., Kajihara, H., Mawatari, S. F., Noguchi, T., Miyazawa, K.(2003) Paralytic toxicity in the ribbon worm Cephalothrix species (Nemertea) in Hiroshima Bay, Hiroshima Prefecture, Japan and the isolation of tetrodotoxin as a main component of its toxins. *Toxicon*, 41:747-753.

**Chapter 11** 

© 2012 Ismaya et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Ismaya et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Chromatography as the Major Tool in** 

**from Saccharomycopsis Fibuligera R64** 

Wangsa T. Ismaya, Khomaini Hasan, Toto Subroto,

Dessy Natalia and Soetijoso Soemitro

also biochemical analysis of the enzymes.

**2.** *S. fibuligera* **R64 secretes amylolytic enzyme** 

attractive workhorse for the bio-ethanol production.

http://dx.doi.org/10.5772/51325

**1. Introduction** 

Additional information is available at the end of the chapter

**the Identification and the Structure-Function** 

Chromatography, at both preparative and analytical levels, has been a key experimental technique in the study of proteins, primarily liquid chromatography in the separation, purification, and analysis. Recent developments in the study of proteins lean towards simplification and miniaturization, thus chromatography becomes less involved and explored. For example, development of protein tags and their associated affinity matrices enables purification of a protein in one step. This chapter describes the identification and structure-function relation study of amylolytic enzymes from *Saccharomycopsis fibuligera* R64, where liquid chromatography was used as the major tool, not only in the purification but

*S. fibuligera* is a food-borne yeast that is widely used in the production of rice- or cassavabased fermented food, i.e. *Tape* in Indonesia and other Southeast Asia countries (1). The yeast, in combination with *Saccharomyces cerevisiae* or *Zymomonas mobilis*, has been employed in the production of ethanol using cassava starch as the starting material (2), where the starch is firstly degraded into simple sugars prior to (bio-) ethanol. Bioethanol has been promoted as a renewable energy replacing fossil fuels and at the moment is already used as an additive. As the demand for renewable energy grows, *S. fibuligera* emerges as an

**Relationship Study of Amylolytic Enzymes** 

