Bacillus cereus TKU034所生產之幾丁聚醣酶的純化與應用

Purification and application of a chitosanase from Bacillus cereus TKU034

駱柏蒼(Bo-Chang Lo)

指導教授 ： 王三郎

淡江大學/理學院/化學學系碩士班/碩士(2014年)

https://doi.org/10.6846/TKU.2014.00486

摘要

菌株Bacillus cereus TKU034係篩選自新北市三芝山區土壤之幾丁聚醣酶及蛋白酶生產菌，以含有1%烏賊軟骨粉、0.1% K2HPO4、0.05% MgSO4．7H2O之100mL液態培養基，於37°C中搖瓶(150 rpm)培養4天可得較高幾丁聚醣酶活性(0.68U)。
依較佳培養條件培養所得之發酵液，經離心、硫酸銨沉澱、DEAE-Sepharose及 Macro-PrepR DEAE Cartridge陰離子交換層析等步驟，以高純化度(441倍)以及高活性回收率(50.9%)，純化出經SDS PAGE電泳測得分子量約為43 kDa之一種幾丁聚醣酶。此幾丁聚醣酶的最適反應pH為7、pH安定性為5~7.5 、最適反應溫度為50°C、於50°C以下皆具熱安定性，其活性會受到Fe2+、Ca2+、Cu2+、Zn2+、Mn2+及EDTA所抑制。
TKU034之發酵上清液或水解WSC之水解上清液具有DPPH自由基清除能力及亞鐵離子螯合能力，且其發酵上清液對格蘭氏陽性菌S. aureus及B. subtilis之生長具有抑制之效果。色素吸附方面，烏賊軟骨粉經過TKU034發酵後對於食用色素之吸附效果提高10%左右，對於分散染料則提高約40%，且吸附後之染料不易再被脫附之性質對於汙水處理方面有著潛力。

關鍵字

Bacillus cereus ； 幾丁聚醣酶 ； 烏賊軟骨

並列摘要

The chitosanase and protease producing strain, TKU034, was isolated from the soil in Sanzhi, Taiwan with squid pen powder (spp) as the sole carbon/nitrogen source and identified as Bacillus cereus.The optimized culture conditions for chitosanase production by B. cereus TKU034 was found when the organism was cultured at 37°C with 150 rpm for 4 days in 100 mL of medium containing 1% SPP、0.1% K2HPO4 and 0.05% MgSO4．7H2O.
The chitosanase (CHS) with 441-fold of purifying rate and 50.9% of activity yield was purified from the culture supernatant of B. cereus TKU034 by ammonium sulfate precipitation, DEAE-Sepharose and Macro-PrepR DEAE Cartridge chromatography. The molecular mass of CHS determined by SDS-PAGE was approximately to be 43 kDa. The optimum pH, pH stability , optimum temperature and thermal stability of CHS was pH 7, pH 5-7.5, 50°C, <50°C. The CHS was inhibited by Fe2+、Ca2+、Cu2+、Zn2+、Mn2+ and EDTA.
The antioxidation activities of culture supernatant of B. cereus TKU034 and hydrolyzates of water soluble chitosan were measured with DPPH radical scavenging capacity and ferrous ion chelating ability.The culture supernatant has inhibitory effect against S. aureus and B. subtilis TKU007.The pigments adsorption capacity of fermented SPP was better than that of unfermented SPP.

並列關鍵字

Bacillus cereus ； chitosanase ； squid pen

參考文獻

黃稚婷 (2014) Bacillus cereus TKU033 所生產幾丁聚醣酶之純化及其應用。私立淡江大學化學所碩士論文。第51-62頁。

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