Serratia marcescens TKU011所生產蛋白酶及幾丁聚醣酶之純化與定性

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Title:	Serratia marcescens TKU011所生產蛋白酶及幾丁聚醣酶之純化與定性
Other Titles:	Purification and characterization of proteases and chitosanase from Serratia marcescens TKU011
Authors:	彭若華;Peng, Jo-hui
Contributors:	淡江大學生命科學研究所碩士班王三郎;Wang, San-lang
Keywords:	Serratia marcescens;蛋白酶;幾丁聚醣酶;蝦殼粉;Serratia marcescens;protease;chitosanase;shrimp shell wastes
Date:	2007
Issue Date:	2010-01-11 02:25:30 (UTC+8)
Abstract:	TKU011係以蝦殼粉為唯一碳/氮源，篩選自台灣中部土壤之一株蛋白酶及幾丁聚醣酶生產菌，經鑑定為Serratia marcescens。TKU011生產蛋白酶和幾丁聚醣酶之較適培養條件為2%蝦殼粉、0.1%K2HPO4及0.05%MgSO4‧7H2O，在25℃、pH7之50mL液態培養基振盪培養2天。發酵所得上清液經硫酸銨沉澱、DEAE-Sepharose及Sephacryl S-100層析步驟後，可分離出一種蛋白酶(P1)及一種幾丁聚醣酶(C1)，經SDS-PAGE測得分子量分別為50kDa及21kDa。利用Mascot資料庫比對，P1胺基酸序列分析結果與Serratia protease有8段胜肽相同，P1之N末端胺基酸序列為：AATTGYDAVDDLLHYHER。其最適反應pH、最適反應溫度、pH安定性、熱安定性分別為pH10，50℃，pH5-11，<40℃；其活性會受Cu2+、EDTA所抑制，而2%(v/v) Tween 20和Triton X-100則完全抑制。C1之胺基酸序列分析經與Mascot資料庫比對，與Chitin-Binding Protein Cbp21有3段胜肽相同。其最適反應pH、最適反應溫度、pH安定性、熱安定性分別為pH5，55℃，pH6-8，<50℃；其活性會受Mn2+、Fe2+、EDTA所抑制，而2%(v/v) Tween 40 提升165%。 The protease and chitosanase producing strain, Serratia marcescens TKU011, was isolated from the soil in Taiwan. The optimized culture condition for production of the protease and chitosanase was composed of 2% shrimp shell powder (SSP), 0.1% K2HPO4, 0.05% MgSO4‧7H2O at pH7 and incubated in 250 mL Erlenmeyer flask containing 50 mL kept shaking at 30℃ for 2 days . The protease and chitosanase were purified from the culture supernatant by chromatography procedures of DEAE-Sepharose, and Sephacryl S-100. The molecular mass of protease and chitosanase determined by SDS-PAGE was approximately 50 kDa and 21 kDa, respectively. According to Mascot there were eight fragments of peptide by the amino acid assay of the protease which were the same with the Serratia protease. The N-terminal amino acid sequence of the protease contains: AATTGYDAVDDLLHYHER. The optimum pH, optimum temperature, pH stability, thermal stability of protease were pH10, 50℃, pH5-11, <40℃, respectively. The protease was inactivated by Cu2+, Fe2+, EDTA, 2% (v/v) Tween 20 and 2% (v/v) Triton X-100. According to Mascot there were three fragments of peptide by the amino acid sequencing of the chitosanase which were the same with the Chitin-Binding Protein Cbp21. The optimum pH, optimum temperature, pH stability, thermal stability of chitosanase were pH5, 60℃, pH6-8, <50℃, respectively. The chitosanase was inactivated by Mn2+, Fe2+, EDTA, but the chitosanase activated by 2% (v/v) Tween 40.
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