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標題: The study on Chitinibacter tainanensis Cultivation and the Application of Its Broth as an Antagonist to Plant Pathogens
探討Chitinibacter tainanensis醱酵及其醱酵液應用於植物病原菌之拮抗測試
作者: 黃功銘
Huang, Gong-Ming
關鍵字: Chitinibacter tainanensis;Chitinibacter tainanensis;chitinase;N-acetyl-glucosaminidase;antagonistic;幾丁質;幾丁質酵素;N-乙醯葡萄糖胺酶;拮抗試驗
出版社: 化學工程學系所
In this study, the fermentation of Chitinibacter tainanensis was carried out to produce chitinase and N-acetyl-glucosaminidase (NAGase) and to evaluate their effects on the antagonistic effect against plant pathogens. It was found that when using yeast extract as the nitrogen sources and calsium carbonate as the buffer salt, the cell mass would reach at 2.54 g/L. Meanwhile, by using chitin as the carbon source, the chitinase and N-acetyl-glucosaminidase would reach 13.2 mU/mL and 558.3 mU/mL in the supernatant of the harvested broth, respectively.
It was observed that the cell precipitate of the broth still reserved chitinase and N-acetyl-glucosaminidase activities. Accordingly, 50 mM Tris buffer (pH7.4) was used to wash out the residual enzymes activities from the cells. The result showed that 7.17 mU/ml of chitinase and 558.3 mU/ml of NAGase can be obtained under a 10-min sonication. It was found that Na2(SO)4 increased the chitinase and NAGase activity for 193.80% and 137.22% respectively, but Cu2+, Ca2+, Mg2+, Zn2+, Co2+, Ni2+ and Mg2+ inhibited NAGase activity.
To test the effect of these collected solutions to the antagonistic effects against plant pathogens, various microorganisms were used in the tests. It was found that the supernatant displayed a significant inhibitive effect on Scherotium rolfsii. Whereas, the washing-out buffer containing enzymes showed no antagonistic effect. This result proved that the antagonistic effect of the broth was due to some unknown chemicals and not from chitinase and N-acetyl-glucosaminidase. Further investigations should be performed to reveal the compounds and mechanisms about this antagonistic effect of the fermentation broth. The supernatant obtained at 48 hour had inhibition ratio of 78%, and that obtained at 24 hour gave an 83% inhibition ratio.

本研究以幾丁質為碳源,使用Chitinibacter tainanensis菌種生產幾丁質酵素與N-乙醯葡萄糖胺酶,將幾丁質分解為幾丁寡醣以及N-乙醯葡萄糖胺。培養基篩選時,以yeast extract為氮源時,菌體濃度可達到 1.88 g/L。因培養過程中pH下降會造成菌體停止生長或死亡,藉由添加碳酸鈣4g/L當做緩衝,將醱酵液pH維持於5以上,菌量可達2.45g/L。使用修飾後含幾丁質之培養基可生產幾丁質酵素 13.2mU/mL、N-乙醯葡萄糖胺酶558.3mU/mL,醱酵液中幾丁質分解N-乙醯葡萄糖胺量為16g/L。
將醱酵液離心所得菌體,經由50 mM Tris-HCl,pH7.4,於超音波震盪10 min ,並去除菌體後,所得上清液中含幾丁質酵素7.17 mU/ml與N-乙醯葡萄糖胺酶558.3 mU/ml,證明菌體沈澱物中帶有幾丁質酵素與N-乙醯葡萄糖胺酶,添加金屬離子嘗試改變酵素活性的表現,結果顯示N-乙醯葡萄醣胺酶活性受到5 mM二價金屬離子,如Cu2+、Ca2+、Mg2+、Zn2+、Co2+、Ni2+及 Mg2+抑制N-乙醯葡萄糖胺酶,幾丁質酵素受金屬離子抑制影響較小,Na2SO4則有助於幾丁質酵素與N-乙醯葡醣胺酶的活性表現,比較控制組幾丁質酵素與N-乙醯葡萄醣胺酶活性,為原來酵素活性之193.80%和137.22%。另外,於EDTA 存在下,酵素活性也提升。
將Chitinibacter tainanensis醱酵培養之上清液,用於農作物拮抗病原菌,發現醱酵液對水稻秧苗立枯病(Sclerotium rolfsii)有明顯之抑菌能力,其效率達78%。經由進一步方法證實,醱酵液中所含之幾丁質酵素、N-乙醯葡萄醣胺酶和糖度皆非產生拮抗之物質。推測應該有其他分子量小於1kDa的物質為其抑菌之主要成分,且此物質經由高溫高壓之操作條件,仍可保有其抑菌活性。
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