Bacillus cereus TKU028 所生產幾丁質酶及蛋白酶之純化、定性與應用

Purification and characterization of a chitinase and a protease from Bacillus cereus TKU028 and their applications

謝東諺(Tung-Yen Hsieh)

指導教授 ： 王三郎

淡江大學/理學院/化學學系碩士班/碩士(2011年)

https://doi.org/10.6846/TKU.2011.00678

摘要

菌株TKU028係以蝦殼廢棄物作為唯一碳/氮源，篩選自彰化土壤之一株幾丁質酶及蛋白酶生產菌，經鑑定為Bacillus cereus。
本研究藉由B. cereus TKU028發酵蝦殼廢棄物生產幾丁質酶及蛋白酶，並探討其較適培養條件。酵素純化係利用酵素與基質專一性之特性進行。B. cereus TKU028所生產幾丁質酶及蛋白酶，經由SDS-PAGE測得分子量分別為40kDa及43kDa，而最適反應pH、最適反應溫度、pH安定性及熱安定性分別為：pH5-6, 60℃, pH5-8, ≦ 60℃；pH9, 50-60℃, pH5-9, ≦ 40℃。幾丁質酶除了受Mn2+及EDTA有約40%抑制外，皆不受金屬離子及界面活性劑所影響；蛋白酶則受到Cu2+、SDS及EDTA抑制，判定屬於金屬型蛋白酶。
B. cereus TKU028之幾丁質酶水解幾丁質所得寡糖，藉由HPLC分析得知以 (GlcNAc) 6為主；而蛋白酶因耐鹼及界面活性劑特性具潛力應用於洗滌劑添加物及去毛劑；PCR-DGGE結果顯示，B. cereus TKU028可與蝦蟹殼廢棄物應用於環境之生物復育。

關鍵字

仙人掌桿菌 ； 幾丁質酶 ； 蛋白酶 ； 蝦頭粉

並列摘要

Bacillus cereus TKU028, a chitinase and protease-producing strain, was isolated from the soil in Taiwan, by using shrimp head powder (SHP) as the sole carbon/nitrogen source. A chitinase (CHT) and a protease (PRO) were purified by chitin affinity binding and casein affinity binding, respectively. The molecular masses of CHT and PRO determined by SDS- PAGE were approximately 40 kDa and 43 kDa, respectively.
The optimum pH, optimum temperature, pH stability, and thermal stability of CHT and PRO were pH 5-6, 60 ℃, pH 5-8, and ≦60 ℃, and pH 9,50-60 ℃, pH 5-9, and ≦ 40℃, respectively. CHT was little inhibited by Mn2+ and EDTA, and PRO was inhibited by Cu2+ , EDTA, and 2 mM SDS.
The chitinase-hydrolyzed products of subtract degradation were analyzed by HPLC at 1-48 h intervals. It was observed the major products are (GlcNAc)6. In addition, B. cereus TKU028 has unlimited potential to enhance the biodegradtion of shrimp shells in the seawater containing mangrove river sediment.

並列關鍵字

Bacillus cereus ； chitinase ； protease ； shrimp head powder

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