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機構典藏 > College of Sciences > Graduate Institue of Life Sciences > Thesis > Item 987654321/32604

Please use this identifier to cite or link to this item: https://tkuir.lib.tku.edu.tw/dspace/handle/987654321/32604

Title:	Chryseobacterium indologenes TKU014所生產三種蛋白酶之純化及定性
Other Titles:	Purification and characterization of three proteases from chryseobacterium indologenes TKU014
Authors:	許菀庭;Hsu, Wan-ting
Contributors:	淡江大學生命科學研究所碩士班王三郎;Wang, San-lang
Keywords:	Chryseobacterium indologenes;蛋白酶;Chryseobacterium indologenes;protease
Date:	2007
Issue Date:	2010-01-11 02:26:09 (UTC+8)
Abstract:	以蝦殼粉為唯一碳/氮源，自台灣土壤篩選到Chryseobacterium indologenes TKU014此株蛋白酶生產菌。 C. indologenes TKU014在含有0.5% 蝦殼粉、0.05 % MgSO4．7H2O及0.1 % K2HPO4(pH6)的液體培養基，於30℃培養一天，可得到較高的蛋白酶活性(0.44U/mL)。所得發酵液之離心上清液經過硫酸銨沉澱、離子交換樹脂層析法及疏水性層析分離等純化步驟，得到三種蛋白酶P1、P2和P3，經SDS-PAGE測得分子量分別為56kDa、40kDa和40kDa。最適反應溫度分別為30~50℃、40℃和40~50℃，最適pH均偏鹼性分別為pH10、pH8和pH9，其pH安定性為pH5~11、pH6~8和pH8~10，熱安定性則為<50℃、<40℃和<40℃。三種蛋白酶的活性均受Mn2+、Cu2+、Fe2+的抑制。P1、P2、P3均受EDTA和1,10-phenanthroline的抑制，判定，P1、P2和P3皆屬Zn-金屬型蛋白酶。基質特異性方面，對酪蛋白、彈性蛋白和角蛋白為基質時，蛋白酶P1、P2和P3具有較佳的活性，對於白蛋白、纖維蛋白、血球蛋白、偶氮白蛋白、偶氮酪蛋白的活性不佳。 Chryseobacterium indologenes TKU014, a protease-producing strain, was isolated from the soil in Taiwan, by using shrimp shell powder (SSP) as the sole carbon/nitrogen source. The optimized conditions for protease production was found when the culture was shaken at 30℃ for one day in 50mL of medium (pH6 ) containing 0.5% SSP, 0.1% K2HPO4, 0.05% MgSO4．7H2O Three proteases (P1, P2, and P3) were purified from culture supernatant by ammonium sulfate precipitation, ionic exchange of DEAE-sepharose CL-6B chromatography and Phenyl Sepharose hydrophobic interaction. The molecular mass of TKU014 proteases (P1, P2, and P3) determined by SDS-PAGE was approximately 56 kDa, 40 kDa, and 40 kDa, respectively. The three proteases (P1, P2, and P3) were found to have optimum temperature at30~50℃,40℃,and40~50℃; optimum pH at 10, 8 and 9; thermal stability lower then 50℃, 40℃ and 40℃ ; pH stability at pH 5~11, pH 6~8 and pH 8~10, respectively. The activity of three proteases (P1, P2, and P3) was completely inactivated by EDTA and 1,10-phenanthroline, so the three proteases (P1, P2, and P3) were Zn-metalloprotease. As for substrate specificity, these three proteases showed good activity toward casein, elastin and keratin azure as substrates, low activity with hemoglobin, and poor activity with albumin, fibrin, hemoglobin, azocasein, albumin.
Appears in Collections:	[Graduate Institue of Life Sciences] Thesis

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