綠竹筍懸浮細胞幾丁質酶cDNA之選殖與檢定

Molecular Cloning and Characterization of Chitinase cDNA from Suspension-Cultured Cells of Green Bamboo (Bambusa Oldhamii)

楊季翰(Ji-Han Yang)

指導教授 ： 宋賢一

共同指導教授 ： 王愛玉

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.01834

摘要

從綠竹筍懸浮細胞 cDNA 庫篩選到4株cDNA，分別命名為 BoCHI3-1a、BoCHI3-1b、BoCHI3-2和BoCHI3-3，這4株cDNA核苷酸序列推衍之胺基酸序列與其他物種之第三型幾丁質酶序列具有極高之相似性，並且含有植物之第三型幾丁質酶的特徵，如6個保守性之Cysteine，以及催化中心之Glutamate，在經過網站 SWISS-MODEL 之電腦程式分析後，發現此4株 cDNA 相對應之蛋白質結構與其他植物第三型幾丁質酶之結構相同，都是由8個 α-helix 與8個β-sheet 折疊而成之桶狀結構。 將 BoCHI3 cDNAs 轉譯區之序列與表現載體pTrcHisB連結後，轉殖到大腸桿菌 TOP10 中進行重組蛋白質表現。在以 IPTG 誘導後，發現在 SDS-PAGE電泳之內切型幾丁質酶活性染色可發現每株轉形株皆有內切型幾丁質酶活性，但是若以還原糖增量法來測定內切型幾丁質酶活性，則只有轉形株 BoCHI3-1as 可測得酵素活性，可能是因為以上兩種活性測定法靈敏度不同，所以造成以上結果的差異。此外，由於重組蛋白質之 N 端含有His tag，因此也利用Ni-NTA親和性層析來進行轉形株 BoCHI3-1as 的純化，結果發現此步驟可以有效的去除大部分的雜蛋白質，達到純化的效果。

關鍵字

幾丁質 ； 幾丁質酶

並列摘要

Four cDNAs, named BoCHI3-1a, BoCHI3-1b, BoCHI3-2 and BoCHI3-3, were cloned from a cDNA library constructed from suspension cells of green bamboo (Bambusa oldhamii). The deduced amino acid sequences of the four cDNAs show high similarity to class III chitinases of other species. The presence of six conserved cysteine residues and a conserved glutamate residue at the putative active site, which are characteristics of class III chitinases in plants, confirms that the four cDNAs encode class III chitinases. The four cDNA encoded proteins all have
（β/ㄐ^8 barrel fold, a typical structure like other class III chitinase from other plants, as revealed by the computer program analysis (SWISS-MODEL). The open reading frame of the four BoCHI3 cDNAs were cloned into the plasmid pTrcHisB, and then transformed into E.coli. TOP10 for expression. By induction with IPTG, endochitinase activity in the enzyme extracts of the transformants could be detected by activity staining on SDS-PAGE. However only the activity of the recombinant BoCHI3-1as could be detected by reducing sugar method. These results suggest the different sensitivity of activity assay methods. Furthermore, the transformants have a His tag at N-terminus hence can be purified by Ni-NTA affinity chromatography. According to the result, this step can really achieve the purpose of the purification.

並列關鍵字

chitin ； chitinase

參考文獻

Abdel-Naby, M. A.., El-Shayeb, N. M. A. & Sherief, A. A. 1992. Purification and some properties of chitinase from Aspergillus Carneus. Appl. Biochem. Biotechnol. 37:141-154 Anke C Terwisscha van Scheltinga, Kor H Kalk, Jaap J Beintema, Bauke W Dijkstra 1994. Crystal structures of hevamine, a plant defence protein with chitinase and lysozyme activity, and its complex with an inhibitor. Structure, Vol 2, 1181-1189 Bade, M. L. & Stinson, A. 1979. Molting fluid chitinase a homotropic allosteric enzyme. Biochem. Biophys. Res. Commun. 87:349-353 Blaiseau, P. L. & Lafay, J.F. 1992. Primary structure of a chitinase- encoding gene (chi 1) from the filamentous fungus Aphanocladium album:similarity to bacterial chitinases. Gene. 120:243-248 Burnette, W. N. 1981. “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A Anal. Biochem. 112:195-203 Chang CT, Lo HF, Wu CJ, Sung HY. 1992. Purification and properties of chitinase from cabbage. Biochem Int. Dec;28(4):707-15 Collinge D. B., Kragh, K. M. 1993. Plant chitinase. Plant J. 3:31-40 Desouza MM, Murray MK. 1995. An estrogen-dependent secretory protein, which shares identity with chitinases, is expressed in a temporally and regionally specific manner in the sheep oviduct at the time of fertilization and embryo development. Endocrinology 136:2485-2496 81

Dickinson, K., Keer, V., Hitchcock, C.A. & Adams, D. J. 1989. Chitinase activity from Candida albicans and its inhibition by allosamidin. J. Gen. Microbiol. 135:1417-1421 Flach, J., Pilet, P.E.& Jolles, P. 1992. What’s new in chitinase research? Experimenta. 48:701-716 Hackamn, R. H. 1960. Studies on chitin. . The occurrence of complⅣexes in which chitin and protein are covalently linked. Aust. J. Biol. Sci. 13:568-579 Hart PJ, Ready MP, Robertus JD. 1992. Crystallization of an endochitinase from Hordeum vulgare L. seeds. J Mol Biol 225:565-567 Hart PJ, Monzingo AF, Ready MP, Ernst SR, Robertus JD. 1993. Crystal structure of an nedochitinase from Hordeum vulgare L. seeds. J Mol Biol 229:189-193 Hart PJ, Pfluger HD, Monzingo AF, Hollis T, Robertus JD. 1995. The refined crystal structure of an endochitinase from Hordeum vulgare L. seeds. at 1.8A resolution. J Mol Biol 248:402-413 Henrissat, B. 1991. A classification of glycosyl hydrolases based on amino acid sequence similarities. J. Biochem. 280:309-316 Hirano, S., Yamamoto, T., Hayashi, M., Nishida, T., Inui, H. 1990. Chitinase activity in seeds coated with chitosan derivates. Agric. Biol. Chem. 54(10):2719-2720 Humphreys AM, Gooday G. 1984. Properties of chitinase activities from Mucor mucedo: evidence for a membrane-bound zymogenic form. J Gen Microbiol 130:1359-1366 Imoto, T. and Yagishita, K. A. 1971 A simple activity measurement of lysozyme. Agric. Biol. Chem. 35: 1154-1156

82

Jekel PA, Hartmann BH, Beintema JJ. 1991. The primary structure of hevamine, an enzyme with lysozyme/chitinase activity from Hevea brasiliensis latex. Eur. J. Biochem. 200:123-130 Jeuniaux, C. 1964. Free chitin and masked chitin in invertebrate skeletal structures. Arch. Int. Physiol. Biochem. 72 (2):329-330 Jeuniaux, C. 1966. Methods in enzymology. Academic Press, New York. Vol. 8, p.644-654 Jeuniaux, C. 1981. Chitinase: an addition to the list of hydrolase in the digestive tract of verterbrates. Nature 192:4798-4799 Ken AB, William A. 1998. Substrate Distortion to a Boat Conformation at Subsite -1 Is Critical in the Mechanism of Family 18 Chitinases. J. Am. Chem. Soc. 120:3571-3580 Koga D. 1996. Comparative biochemistry of insect and plant chitinase. In:RAA Muzzarelli(ed): chitin enzymology, vol 2. Atec, Grottammare, 85-94 Koga D, Sasaki Y, Uchiumi Y, Hirai N, Arakane Y, Nagamatsu Y. 1997. Purification and characterization of Bombyx mori chitinase. Insect Biochem Mol Biol 27:759-767 Kramer KJ, Dziadik-Turner C, Koga D. 1985. Chitin metabolism in insects. In:GA Kerkut, LI Gilbert (eds):Comprehensive insect physiology, biochemistry and pharmacology, vol 3. Integument, respiration and circulation. Pergamon Press, Oxford, 75-115 Kramer KJ, Koga D. 1986. Insect chitin: physical state, synthesis degradation and metabolic regulation. Insect Biochem 16:851-877 Mathivanan N, Kabilan V, Murugesan K. 1998. Purification, characterization, and antifungal activity of chitinase from Fusarium chlamydosporum, a mycoparasite to groundnut rust, Puccinia arachidis. Can J Microbiol. Jul;44(7):646-51

83