Bacillus cereus TKU018所生產幾丁聚醣酶之純化與定性

Purification and Characterization of Chitosanase from Bacillus cereus TKU018

陳姿蓉(Tz-Rung Chen)

指導教授 ： 王三郎

淡江大學/理學院/生命科學研究所碩士班/碩士(2008年)

https://doi.org/10.6846/TKU.2008.00799

摘要

TKU018 係以蝦殼粉為唯一碳/氮源，篩選自台灣北部土壤之幾丁聚醣酶生產菌，經鑑定為Bacillus cereus。TKU018生產幾丁聚醣酶之較適培養條件為含有0.5% 蝦殼粉、0.1 % K2HPO4及0.05 %MgSO4．7H2O之100 mL液態培養基 (pH 9)於37℃下搖瓶培養3天。所得發酵上清液，經硫酸銨沉澱、DEAE-Sepharose、Phenyl-Sepharose等層析步驟，純化出二種幾丁聚醣酶(C1、C2) ，經SDS-PAGE測其C1、C2分子量分別為44 kDa及22 kDa。C1、C2之最適反應溫度、最適反應pH、熱安定性、pH安定性分別為 (60℃、pH 5、<40℃、pH 5~7)，(50℃、pH 7、<50℃、pH 4~7)。C1其活性受Zn²+所抑制，C2則不受影響，在SDS存在下會完全抑制C1，而 C1、C2在0.5% Tween 20存在下，分別還維持原活性之100%及105%。而用烏賊軟骨粉在不同濃度下發酵所得上清液，第三天具有較佳的DPPH自由基清除力。

關鍵字：Bacillus cereus；幾丁聚醣酶；蝦殼粉

關鍵字

幾丁聚醣酶 ； 蝦殼粉 ； Bacillus cereus

並列摘要

The chitosanase producing strain, Bacillus cereus TKU018, was isolated from the soil in the northern Taiwan. The optimized condition for chitosanase production were found when the culture was shaken at 37℃for 3 days in 100mL of medium contain 0.5% shrimp shell powder (SSP), 0.1 % K2HPO4 and 0.05 % MgSO4．7H2O (pH9). Two chitosanases (C1、C2) were purified by chromatography procedures of DEAE-Sepharose, Phenyl-Sepharose. The molecular mass of the C1 and C2 determined by SDS-PAGE was approximately 44 kDa and 22 kDa, respectively. C1、C2 The optimum temperature, optimum pH, thermal stability of C1 and C2 were (60℃、pH 5、<40℃、pH 5~7), (50℃、pH 7、<50℃、pH 4~7), respectively. The C1 was inactivated by Zn²+ , while the C2 was not affected；In the presence of SDS, the C1 was inactivated. C1、C2 retained 100% and 105% of its original activity in the presence of 0.5% Tween 20, respectively.
When cultured on various different concentrations of squid pen powder (SPP), the supernatant of third day have better DPPH radical scavenging activity.

並列關鍵字

Bacillus cereus ； chitosanase ； shrimp shell powder

參考文獻

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