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Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/52091
標題: 在枯草桿菌及地衣芽孢桿菌宿主表現及純化重組幾丁聚醣酶及特性探討
Expression, purification and characteristics of recombinant chitosanases by Bacillus subtilis and Bacillus licheniformis
作者: 詹文嘉
Chan, Wen-Chia
關鍵字: Bacillus licheniformis;幾丁聚醣酶error-prone PCR;chitosanase;error-prone PCR
出版社: 食品暨應用生物科技學系所
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DNA polymerase fidelity and the Polymerase chain reaction. PCR Methods Appl, 1(1), 17-24. Fukamizo, T., Honda, Y., Goto, S., Boucher, I., and Brzezinski, R. (1995). Reaction mechanism of chitosanase from Streptomyces sp. N174. Biochem J, 311 ( Pt 2),377-383. Fukamizo, T., Ohkawa, T., Ikeda, Y., and Goto, S. (1994). Specificity of chitosanase from Bacillus pumilus. Biochim Biophys Acta, 1205(2), 183-188. Gelfand, D. H. and White, T. J. (1990). Thermostable DNA polymerase. In PCR protocols: A guide to methods and applications (ed. M.A. Innis et al.), pp.129-141. Academic Press, San Diego, California, USA. Gupta, R., Beg, Q. K., and Lorenz, P. (2002). Bacterial alkaline proteases: molecular approaches and industrial applications. Appl Microbiol Biotechnol, 59(1), 15-32. Izume, M., Nagae, S., Kawagishi, H., Mitsutomi, M., and Ohtakara, A. (1992). Action pattern of Bacillus sp. no. 7-M chitosanase on partially N-acetylated chitosan. Biosci Biotechnol Biochem, 56(3), 448-453. Jean-François Fiset, Jean-François Blais, Patricio A. Riveros (2008). Review on the removal of metal ions from effluents using seaweeds, alginate derivativesand other sorbents, 21(3), 283-308. Jensen, K. K., and Hulett, F. M. (1989). Protoplast transformation of Bacillus licheniformis MC14. J Gen Microbiol, 135(8), 2283-2287. Jo, Y. Y., Jo, K. J., Jin, Y. L., Kim, K. Y., Shim, J. H., Kim, Y. W., et al. (2003). Characterization and kinetics of 45 kDa chitosanase from Bacillus sp. P16. Biosci Biotechnol Biochem, 67(9), 1875-1882. Kim, K. W., Thomas, R. L., Lee, C., and Park, H. J. (2003). Antimicrobial activity of native chitosan, degraded chitosan, and O-carboxymethylated chitosan. J Food Prot, 66(8), 1495-1498. Ling Lin, F., Zi Rong, X., Wei Fen, L., Jiang Bing, S., Ping, L., and Chun Xia, H. (2007). Protein secretion pathways in Bacillus subtilis: implication for optimization of heterologous protein secretion. Biotechnol Adv, 25(1), 1-12. Ling, L. L., Keohavong, P., Dias, C., and Thilly, W. G. (1991). Optimization of the polymerase chain reaction with regard to fidelity: modified T7, Taq, and vent DNA polymerases. PCR Methods Appl, 1(1), 63-69. Marcotte, E. M., Monzingo, A. F., Ernst, S. R., Brzezinski, R., and Robertus, J. D. (1996). X-ray structure of an anti-fungal chitosanase from streptomyces N174. Nat Struct Biol, 3(2), 155-162. Masson, J. Y., Boucher, I., Neugebauer, W. A., Ramotar, D., and Brzezinski, R. (1995). A new chitosanase gene from a Nocardioides sp. is a third member of glycosyl hydrolase family 46. Microbiology, 141 ( Pt 10), 2629-2635. Rey, M. W., Ramaiya, P., Nelson, B. A., Brody-Karpin, S. D., Zaretsky, E. J., Tang, M., et al. (2004). Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species. Genome Biol, 5(10), R77. Saito, J., Kita, A., Higuchi, Y., Nagata, Y., Ando, A., and Miki, K. (1999). Crystal structure of chitosanase from Bacillus circulans MH-K1 at 1.6-A resolution and its substrate recognition mechanism. J Biol Chem, 274(43), 30818-30825. Schallmey, M., Singh, A., and Ward, O. P. (2004). Developments in the use of Bacillus species for industrial production. Can J Microbiol, 50(1), 1-17. Shih, I. L., and Van, Y. T. (2001). The production of poly-(gamma-glutamic acid) from microorganisms and its various applications. Bioresour Technol, 79(3), 207-225. Tanabe, T., Morinaga, K., Fukamizo, T., and Mitsutomi, M. (2003). Novel chitosanase from Streptomyces griseus HUT 6037 with transglycosylation activity. Biosci Biotechnol Biochem, 67(2), 354-364. Tjalsma, H., Bolhuis, A., Jongbloed, J. D., Bron, S., and van Dijl, J. M. (2000). Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome. Microbiol Mol Biol Rev, 64(3), 515-547. Veith, B., Herzberg, C., Steckel, S., Feesche, J., Maurer, K. H., Ehrenreich, P., et al. (2004). The complete genome sequence of Bacillus licheniformis DSM13, an organism with great industrial potential. J Mol Microbiol Biotechnol, 7(4), 204-211. Waites, W. M., Kay, D., Dawes, I. W., Wood, D. A., Warren, S. C., and Mandelstam, J. (1970). Sporulation in Bacillus subtilis. Correlation of biochemical events with morphological changes in asporogenous mutants. Biochem J, 118(4), 667-676. Young, D. H., and Kauss, H. (1983). Release of calcium from suspension-cultured glycine max cells by chitosan, other polycations, and polyamines in relation to effects on membrane permeability. Plant Physiol, 73(3), 698-702.
摘要: 
枯草桿菌（Bacillos subtilis）與地衣芽孢桿菌（Bacillus licheniformis）皆為革蘭氏陽性（gram-positive）、兼性厭氧（facultative anaerobe）之產孢桿菌，並且都經FDA（Food and Drug Administration；美國食品暨藥物管理局）認可為GRAS（generally recognized as safe）級之宿主，而且具有分泌蛋白質之能力，因此，作為蛋白質分泌宿主於酵素工業之運用上是十分具有發展潛力的。幾丁質（chitin）經由去乙醯化而獲得幾丁聚醣（chitosan），而幾丁聚醣經由幾丁聚醣酶分解後可產生具有多種生理活性之幾丁寡醣（chitooligosaccharide）。幾丁寡醣之生理活性有抑制細菌和真菌生長、增強免疫力、具抗腫瘤活性以及在高等植物中誘導抗菌素的產生。

本實驗室先前已建立枯草桿菌與地衣芽孢桿菌之持續表現系統，利用此系統來表現源自於枯草桿菌之重組幾丁聚醣酶。於本研究中，我們建立了重組幾丁聚醣酶的純化條件以及酵素特性的確立，並且利用隨機突變的方式來改造重組幾丁聚醣酶。實驗中以枯草桿菌與地衣芽孢桿菌作為表現重組幾丁聚醣酶之宿主，兩轉形菌株皆於12小時達到蛋白質最佳表現量，而且地衣芽孢桿菌產量明顯高於枯草桿菌。酵素之特性分析方面，枯草桿菌與地衣芽孢桿菌重組幾丁聚醣酶之最適作用溫度，分別為45℃與40℃；熱穩定性Tm值分別為44℃與46℃；最適作用pH值則分別是5.0與5.5，而兩轉形菌株皆具有大範圍pH值耐受性。酵素動力學分析結果，枯草桿菌重組幾丁聚醣酶的Vmax值為1.578 umole/sec/mg；Km值為3.1×102 mg/ml；kcat/Km值為14.6×10-2 ml/mg/sec。地衣芽孢桿菌重組幾丁聚醣酶的Vmax值為19.27 umole/sec/mg；Km值為25.6×102 mg/ml；kcat/Km值為21.6×10-2 ml/mg/sec。金屬離子Mg2+、Mn2+、Ca2可提升活性，其中又以Mn2+最為明顯，而Fe3+則明顯抑制酵素活性。

本實驗利用error-prone PCR對未含有訊息胜肽基因之幾丁聚醣酶基因進行突變，獲得一突變轉形菌株EP70，基因序列611由guanine突變為adenine（G611A），蛋白質序列194由aspartic acid突變為asparagine（D194N）。

Bacillus subtilis and Bacillus licheniformis are gram-positive, facultative anaerobic endospore-forming bacteria. They have been classified as GRAS (generally recognized as safe) microorganisms by FDA, and have the capability to secret protein. These strains were therefore considered potent hosts to express and secret heterologous proteins. Chitosan is a product obtained by the deacetylation of chitin. Chitosanase hydrolysis of chitosan could be used for preparation of chitosan oligosaccharides which have variety of biological activities, such as inhibiting the growth of bacteria and fungi, acting as immunopotential effectors, exerting antitumor activity, and inducing the phytoalexin product in higher plants.

A constitutive secretory expression system for B. subtilis and B. licheniformis has been established in our laboratory, and express recombinant chitosanase gene originated from B. subtilis DB104. In this study, the recombinant chitosanase secreted by B. subtilis and B.licheniformis purified and analyzed the characteristics. Both transformants expressed highest recombinant chitosanase at 12 hours of culture, and the higher recombinant chitosanase activities secreted by B. licheniformis than B. subtilis. The recombinant chitosanase were purified and the optimal temperature of purified recombinant chitosanase of B. subtilis and B. licheniformis were 45℃ and 40℃ respectively, and the thermostabilities were Tm 44℃ and 46℃. The optimal pH were 5.0 and 5.5 for B. subtilis and B. licheniformis respectively, and the pH stability of recombinant chitosanase were both stable in pH 2.0 to 8.5. The kinetics of recombinant chitosanase by B. subtilis were Vmax of 1.578 umole/sec/mg;Km of 3.1×102 mg/ml;kcat/Km of 14.6×10-2 ml/mg/sec, and recombinant chitosanase secreted by B. licheniformis were Vmax of 19.27 umole/sec/mg;Km of 25.6×102 mg/ml;kcat/Km of 21.6×10-2 ml/mg/sec. The enzyme activities were increased by Mg2+, Mn2+, Ca2+, especially Mn2+.. And Fe3+ significantly inhibited the activity.

In this study, mutated recombinant chitosanase gene obtained by error-prone PCR and got a mutant enzyme EP70. The guanine 611 of wild type chitosanase gene was replaced by adenine (G611A), and the amino acid aspartic acid 194 of wild type chitosanase was replaced by asparagine (D194N).
URI: http://hdl.handle.net/11455/52091
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