Bacillus cereus TKU030 所生產幾丁聚醣酶之純化定性及抑制劑探討

Studies on the purification and characterization of a Bacillus cereus TKU030 chitosanase and its inhibitor

陳岳吟(Yue-Yin Chen)

指導教授 ： 王三郎

淡江大學/理學院/化學學系碩士班/碩士(2012年)

https://doi.org/10.6846/TKU.2012.00704

摘要

Bacillus cereus TKU030 係以烏賊軟骨為唯一碳/氮源，篩選自淡江大學
校園土壤之幾丁聚醣酶、幾丁質酶及蛋白酶之生產菌，本研究探討B. cereus
TKU030 發酵烏賊軟骨生產幾丁聚醣酶之較適培養條件，所得發酵上清液經硫
酸銨沉澱、Sephacryl S-100 膠體層析、DEAE-Sepharose 離子交換層析以及Sephacryl S-100 再膠體層析等步驟，純化出一種幾丁聚醣酶 (CHS)。CHS
之分子量經SDS-PAGE 測為43 kDa；最適反應溫度、最適反應pH、熱安定
性以及pH 安定性分別為40oC、pH 4、30-40oC 以及pH 3-7。於抑制劑方面，
CHS 活性會受Mn2+、Cu2+、Fe2+ 及含硼化合物TKUPSP017 所抑制。

TKUPSP017 濃度於0.05% (w/v) 時的抑制率達100%，抑制效果會隨
CHS 與TKUPSP017 預反應時間的增長而有所提升。此外，抑制劑結構對於
CHS 酵素活性的影響，以及添加TKUPSP017 於培養基對於B. cereus
TKU030 生長之影響亦有探討。CHS 被抑制不會影響TKU030 的生長，隨著
TKUPSP017 濃度提高，TKU030 生長狀況反而越好。

關鍵字

Bacillus cereus ； 幾丁聚醣酶 ； 烏賊軟骨 ； 抑制劑

並列摘要

The chitosanase, chitinase and proease-producing strain
TKU030 was isolated from soil in Tamkang University with squid pen powder as the sole carbon/nitrogen source and identified as Bacillus cereus. A chitosanase (CHS) was purified from the culture supernatant by ammonium sulfate precipitation, Sephacryl S-100 gel chromatography, DEAE- Sepharose column chromatography and Sephacryl S-100 rechromatography . The molecular mass of CHS determined by SDS-PAGE was approximately 43 kDa. The optimum temperature, optimum pH, thermal stability, and pH stability of CHS were 40oC, pH 4, 30-40oC and pH 3-7, respectively.The activity of CHS was inhibited by Mn2+, Cu2+, Fe2+ and a boron-containing compound of TKUPSP017.

The inhibition of CHS activity reached 100% at 0.05% (w/v) of TKUPSP017. The inhibitory activity increased with the
preincubating time of TKUPSP017 and CHS. The effects of
inhibitor structures on enzyme activity and growth of strain TKU030 in media supplemented with TKUPSP017 were also studied.Growth of strain TKU030 was not influenced by inhibiting CHS activity but enhanced by increasing TKUPSP017 concentration.

並列關鍵字

Bacillus cereus ； chitosanase ； squid pen ； inhibitor

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