- 學位論文
Pseudomonas sp. TKU015所生產幾丁質酶、幾丁聚醣酶及納豆激酶之純化與定性
Purification and Characterization of a Chitinase, a Chitosanase, and a Nattokinase from Pseudomonas sp. TKU015
摘要
TKU015 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及幾丁聚醣酶生產菌，經鑑定為Pseudomonas sp.。TKU015生產幾丁質酶及幾丁聚醣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 8)在30℃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，純化出一種幾丁質酶及一種幾丁聚醣酶，經SDS-PAGE測其分子量分別為68 kDa及30 kDa。於最適反應pH、最適反應溫度、pH安定、熱安定性方面，幾丁質酶的為pH 5、50℃、pH 5-7及<60℃；幾丁聚醣酶的為pH 4、50℃、pH 3-9及<50℃。幾丁質酶活性受Mn2+、Fe2+所抑制，幾丁聚醣酶活性則受Mn2+、Cu2+、PMSF所抑制。 TKU015生產納豆激酶之較適生產條件為含有1% 蝦殼粉、0.1 %K2HPO4及0.05 %MgSO4．7H2O 之液態培養基 (pH 7) 於30℃搖瓶培養2天。所得發酵上清液，經硫酸銨沉澱、DEAE-Sepharose及Phenyl-Sepharose等層析分離步驟，純化出一種納豆激酶。經SDS-PAGE測其分子量為21 kDa，其最適反應pH、最適反應溫度、pH安定、熱安定性則分別為pH 7、50℃、pH 4-11及<37℃。納豆激酶活性受PMSF完全抑制，Fe2+則會提高其活性。
並列摘要
The chitinase and chitosanase producing strain, Pseudomonas sp. TKU015, was isolated from the soil in Taiwan. The optimized condition for chitinase and chitosanase production were found when the culture was shaken at 30℃for 3 days in 100mL of medium contain 0.5% shrimp shell powder (SSP), 0.1 % K2HPO4 and 0.05 % MgSO4．7H2O (pH8). One chitinase and one chitosanase were purified by chromatography procedures of DEAE-Sepharose, Phenyl-Sepharose, and Sephacryl S-100. The molecular mass of the chitinase and the chitosanase determined by SDS-PAGE was approximately 68 kDa and 30 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of chitinase were pH 5, 50℃, pH 4-6 and <60℃, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of chitosanase were pH 4, 50℃, pH 3-9 and <50℃, respectively. The chitinase was inactivated by Mn2+ and Fe2+；The chitosanase was inactivated by Mn2+, Cu2+ and PMSF. One nattokinase was also purified from the second day culture supernatant of Pseudomonas sp. TKU015. The optimized condition for nattokinase production was found when the culture was shaken at 30℃for 2 days in 100mL of medium contain 1% SSP, 0.1 % K2HPO4 and 0.05 % MgSO4．7H2O (pH7). The molecular mass of the nattokinase determined by SDS-PAGE was approximately 21 kDa. The optimum pH, optimum temperature, pH stability, and thermal stability of nattokinase were pH 7, 50℃, pH 4-11 and <37℃, respectively. The nattokinase was inactivated by PMSF, and activated by Fe2+.