Serratia sp. TKU019所生產蛋白酶及幾丁聚醣酶之純化與定性

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Title:	Serratia sp. TKU019所生產蛋白酶及幾丁聚醣酶之純化與定性
Other Titles:	Purification and characterization of protease and chitosanase from serratia sp.TKU019
Authors:	郭懿萱;Kuo, Yi-hsuan
Contributors:	淡江大學生命科學研究所碩士班王三郎;Wang, San-lang
Keywords:	幾丁質酶;幾丁聚醣酶;蛋白酶;chitinase;chitosanase;protease
Date:	2008
Issue Date:	2010-01-11 02:28:46 (UTC+8)
Abstract:	TKU019 係以蝦殼粉為唯一碳/氮源，篩選自台灣北部土壤之一株幾丁質酶、幾丁聚醣酶及蛋白酶生產菌，經鑑定為Serratia sp.。TKU019生產幾丁質酶、幾丁聚醣酶及蛋白酶之較適培養條件為含有0.5% 蝦殼粉、0.1 % K2HPO4及0.05 % MgSO4．7H2O之液態培養基 (pH 7)在37℃搖瓶培養3天。所得發酵上清液，經硫酸銨沉澱、DEAE-Sepharose、Macro- prep DEAE Cartridge及Sephacryl S-100等層析步驟，純化出一種幾丁質酶、幾丁聚醣酶 (C1)，及一種蛋白酶 (P1) ，經SDS-PAGE測其分子量分別為36 kDa及58 kDa。於最適反應pH、最適反應溫度、pH安定、熱安定性方面，幾丁質酶為pH 11、60℃、pH 7-10及<40℃；幾丁聚醣酶為pH 7、60℃、pH 4-7及<40℃。蛋白酶之最適反應pH、最適反應溫度、pH安定、熱安定性則分別為pH10、50℃、pH 5-9及<40℃。C1及P1之活性會受Mn2+、EDTA 所抑制，而 2% (v/v) Tween 40可使P1活性提升 175%。探討以烏賊軟骨粉 (SPP)及蝦殼粉 (SSP)作為碳源時，其上清液還原醣量的差異。發現以1.5% SPP及 1% SSP 作為碳源時、發酵第五天及第三天，上清液所含還原醣量最高，分別為 900μg/ml 及 270μg/ml。以SPP及SSP作為碳源時，分析培養上清液的抗氧化活性。結果發現，0.5%及1% 的SPP及SSP，發酵第一天到第四天，皆具DPPH清除效果。 The protease and chitosanase producing strain, Serratia sp. TKU019, was isolated from the soil in Taiwan. The optimized condition for protease and 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 (pH7). One chitosanase (C1) and one protease (P1) were purified by chromatography procedures of DEAE-Sepharose, Macro-prep DEAE Cartridge, and Sephacryl S-100. The molecular mass of the chitosanase (C1) and the protease (P1) determined by SDS-PAGE was approximately 36 kDa and 57 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of C1 were ( pH 11, 60℃, pH 7-10 and <40℃),(using colloidal chitin as substrate) and ( pH7, 60℃, pH 4-7 and <40℃) ,(using chitosan as substrate).Those of P1 were pH 10, 50℃, pH 5-9 and <40℃,respectively. Both of C1 and P1 were inactivated by Mn2+ and EDTA, but only P1 activated by 2% (v/v) Tween 40. The result of the effects of SPP and SSP on reducing sugars show that, the culture supernatant of SPP (1.5%, 5th day) and SSP (1%, 3rd day) had the highest concentration of reducing sugars ( 900 μg/ml and 270 μg/ml, respectively). To analyze antioxidant activity, it was found the culture supernatant of SPP (0.5%, 2 nd day) and SSP (1.5%, 1st day) had the best results of the DPPH free radical scavenging activity.
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