Serratia sp. TKU020所生產蛋白酶、幾丁質酶及幾丁聚醣酶之純化與定性

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题名:	Serratia sp. TKU020所生產蛋白酶、幾丁質酶及幾丁聚醣酶之純化與定性
其它题名:	Purification and characterization of protease, chitinase and chitosanase from serratia sp. TKU020
作者:	劉貞儀;Liou, Jen-yi
贡献者:	淡江大學生命科學研究所碩士班王三郎;Wang, San-lang
关键词:	蛋白酶;幾丁質酶;幾丁聚醣酶;Serratia sp.;protease;chitinase;chitosanase
日期:	2008
上传时间:	2010-01-11 02:29:22 (UTC+8)
摘要:	本研究主要由台灣北部土壤篩選出一株蛋白酶、幾丁質酶及幾丁聚醣酶生產菌TKU020 ，以烏賊軟骨粉為唯一碳/氮源，經鑑定為Serratia sp.。TKU020生產蛋白酶、幾丁質酶及幾丁聚醣酶之較適培養條件為0.5% 烏賊軟骨粉、0.1 % K2HPO4及0.05 %MgSO4．7H2O，在25℃、pH 9之100 mL液態培養基搖瓶培養3天。所得發酵上清液經硫酸銨沉澱、DEAE-Sepharose、Phenyl- Sepharose及Sephacryl S-100等層析步驟，純化出一種蛋白酶、一種幾丁質酶及一種幾丁聚醣酶，經SDS-PAGE測其分子量分別為55.4 kDa、64.5 kDa及55.4 kDa。其最適反應溫度、熱安定性、最適反應pH、pH安定方面，蛋白酶的分別為50℃、40℃、pH 10及pH 4-11；幾丁質酶為40℃、30℃、pH 4-8及pH 4-8；幾丁聚醣酶為40℃、40℃、pH 5及pH 7。蛋白酶活性不受金屬離子、EDTA、PMSF所抑制，幾丁質酶和幾丁聚醣酶皆受Mn2+、SDS所抑制。利用TKU020這株菌，分別用不同碳/氮源 (0~2%的蝦殼粉和烏賊軟骨粉)培養在25℃、pH 9之100 mL液態培養基分別培養0~5天，所得發酵上清液去探討其還原糖量和抗氧化之能力。 Strain TKU020 was isolated from the soil in the northern Taiwan by using squid pen powder (SPP) as the sole carbon/nitrogen source and identified as Serratia sp. .The optimized culture condition for protease, chitinase and chitosanase was composed of 0.5% squid pen powder (SPP), 0.1 % K2HPO4 and 0.05 % MgSO4．7H2O at pH 9 and incubate in 100 mL of liquid media in shaking flasks at 25℃ for 3 days. One protease, chitinase and chitosanase were purified by chromatography procedures of DEAE-Sepharose, Phenyl-Sepharose, and Sephacryl S-100. The molecular mass of protease, chitinase and chitosanase determined by SDS-PAGE was approximately 49 kDa, 55.4 kDa and 65.4 kDa, respectively. The optimum temperature, thermal stability, optimum pH and pH stability of protease were 50℃, 40℃, pH 10 and pH 4-11, respectively. The optimum temperature, thermal stability, optimum pH and pH stability of chitinase were 40℃, 30℃, pH 4-8 and pH 4-8, respectively. The optimum temperature, thermal stability, optimum pH and pH stability of chitosanase were 40℃, 40℃, pH 5 and pH 7, respectively. The protease activity was not inhibited by tested metal ions, EDTA and PMSF；The chitinase and chitosanase were both inactivated by Mn2+ and SDS. In the second part , Serratia sp. TKU020 was cultured using different carbon source (0~2% shrimp shell powder and squid pen powder)and incubated in 100 mL of liquid media (pH 9) in shaking flasks at 25℃ for 0~5days , and the quantity of DPPH scavenging activity and reducing sugar in culture supernatant was studied.
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