Bacillus subtilis TKU007所生產幾丁聚醣酶及耐有機溶劑蛋白酶之純化與定性

Purification and Characterization of a Chitosanase and a Solvent Stable Protease from Bacillus subtilis TKU007

葉佩怡(Pei-Yi Yeh)

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(2006年)

https://doi.org/10.6846/TKU.2006.00127

摘要

Bacillus subtilis TKU007係株以蝦殼粉為主要碳源，篩選自台灣北部土壤之蛋白酶生產菌，發酵蝦殼粉所得離心上清液具有幾丁聚醣酶及蛋白酶活性。幾丁聚醣酶較適生產條件為1.5% 蝦殼粉、0.1%
K2HPO4、0.05% MgSO4．7H2O之液態培養基(pH7)於30℃振盪培養1天。發酵所得離心上清液經硫酸銨沉澱、DEAE-Sepharose及Sephacryl-S100管柱層析後，可純化出幾丁聚醣酶。經SDS-PAGE及膠體層析測定分子量分別為25 kDa及33 kDa。TKU007幾丁聚醣酶之N末端胺基酸序列含：PQNI。其最適反應pH、最適反應溫度、pH安定性、熱安定性、Km 及Vmax分別為pH 7, 37℃, pH 4-9,25-37 ℃, 0.438 mg/mL, 0.144 U/mL；其活性會受Cu2+所抑制，而2%(v/v) Tween 40或Triton X-100則不具抑制效果。
B. subtilis TKU007生產耐界面活性劑、耐有機溶劑及對鹼性安定性高之絲胺酸蛋白酶之較適培養條件為1.5% 蝦殼粉、0.1% K2HPO4
、0.05% MgSO4．7H2O之液態培養基(pH7)於30 ℃培養2天。發酵所得離心上清液離心，經硫酸銨沉澱、DEAE-Sepharose、Phenyl Sepharose及Sephacryl S-100管柱層析後，純化出一分子量經SDS-PAGE及膠體層析測定為28 kDa及30 kDa之蛋白酶，且培養第一天時發現TKU007蛋白酶會造成幾丁聚醣酶活性降低。TKU007蛋白酶之N末端胺基酸序列為:AQSVPYGISQIKAPALGSQG。其最適反應pH、最適反應溫度、pH安定性、熱安定性、Km 及Vmax分別為pH 7-11, 50 ℃, pH 5-11, 50 ℃, 0.13 mg/mL, 0.86 U/mL；在25%有機溶劑存在下，於25℃及4℃放置10天，蛋白酶仍維持原活性80%以上。此外，蛋白酶在2% Tween 20、2% Tween 40及2 mM SDS存在下分別還維持原活性之100%、100%及69%。

關鍵字

Bacillus subtilis ； 耐鹼性蛋白酶 ； 界面活性劑安定 ； 有機溶劑安定 ； 蝦殼粉 ； 幾丁聚醣酶

並列摘要

The chitosanase and protease-producing bacterium, Bacillus subtilis TKU007, was isolated from soil in the north Taiwan. The supernatant of the culture medium contains the protease and the chitosanase activity. The optimized culture condition for production of the chitosanase was composed of 1.5% SSP,0.1% K2HPO4,0.05% MgSO4．7H2O at pH 7 and incubation in 250mL Erlenmeyer flask containing 200mL kept shaking at 30℃for one day. The chitosanase was purified from the culture supernatant by ammonium sulfate precipitation, DEAE-Sepharose, and Sephacryl-S100. The molecular mass of TKU007 chitosanase determined by SDS-PAGE and gel filtration was approximately 25 kDa and 33 kDa, respectively. The N-terminal amino acid sequence of the protease contains PQNI. The optimum pH, optimum temperature, pH stability, thermal stability, Km, and Vmax of TKU007 chitosanase were pH7, 37℃, pH4-9, 25-37℃, 0.438 mg/mL, and 0.144 U/mL, respectively. TKU007 chitosanase was inactivated by Cu2+,but not by 2%Tween 40, and 2%Triton X-100.
An extracellular serine protease with novel properties of surfactant-
stable, solvent-stable, and alkaliphilic was purified from B. subtilis TKU007. The optimized culture condition for production of the protease was composed of 1.5% SSP,0.1% K2HPO4, 0.05% MgSO4．7H2O at pH 7 and incubation in 250mL Erlenmeyer flask containing 100mL kept shaking at 30℃ for two days. The protease was purified from the culture supernatant by ammonium sulfate precipitation, DEAE-Sepharose, Phenyl Sepharose, and Sephacryl-S100. The molecular mass of TKU007 protease determined by SDS-PAGE and gel filtration was approximately 28 kDa and 30 kDa, respectively. The TKU007 protease showed suppressing effect on the chitosanase, which was apparent at the first day. The N-terminal amino acid sequence of the protease was: AQSVPYGISQIKAPALGSQG. The optimum pH, optimum temperature, pH stability, thermal stability, Km, and Vmax of TKU007 protease were pH 7-11, 50℃, pH 5-11, 50℃, 0.13 mg/mL, and 0.86 U/mL, respectively. More than 80% of the original activity was retained even after preincubation for 10 days at 25℃or 4℃ in the presence of 25% tested organic solvents. Additionally, the TKU007 protease retained 100%, 100%, and 69% of its original activity in the presence of 2% Tween 20, 2% Tween 40, or 2 mM SDS, respectively.

並列關鍵字

Bacillus subtilis ； alkaliphilic protease ； surfactent stable ； solvent stable ； shrimp shell wastes ； chitosanase

參考文獻

11. 蘇遠志，黃世佑 (2002) 微生物化學工程學。第372 - 374頁。

5. 莊榮輝 (2000) 酵素化學實驗。國立台灣大學農業化學系生物化

6. 張雅敏 (2002) 黑豆種子幾丁質酶之純化與性質研究：第39-43

12. Adinarayana, K., Ellaiah, P. and Prasad, D. S.

Purification and partial characterization of thermostable serine alkaline protease from a newly isolated Bacillus subtilis PE-11. AAPS Pharm. Sci.Tech. 2003；4：56.