木瓜酵素所含蛋白酶及幾丁質酶之純化與定性

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Title:	木瓜酵素所含蛋白酶及幾丁質酶之純化與定性
Other Titles:	Purification and characterization of proteases and chitinases from papain
Authors:	呂宜倩;Lu, Yi-chien
Contributors:	淡江大學生命科學研究所碩士班王三郎;Wang, San-lang
Keywords:	木瓜酵素;蛋白酶;幾丁質酶;papain;protease;chitinase
Date:	2006
Issue Date:	2010-01-11 02:25:03 (UTC+8)
Abstract:	市售木瓜酵素經由DEAE-Sepharose、CM-Sepharose、Sephacryl S-100管柱層析後，可獲得兩種具有幾丁質酶活性的蛋白酶，分子量分別為26kDa（F1）及28kDa（F2）。以酪蛋白為基質，F1和F2最適反應溫度、pH、熱安定性及pH安定性分別為（70℃、7、40-60℃、5-11）和（80℃、8、25-60℃、4-10）；以懸浮態幾丁質為基質，F1和F2最適反應溫度、pH、熱安定性及pH安定性分別為（50℃、4、25-50℃、5-8）和（40℃、4、30-70℃、4-7）。金屬離子對酵素影響，以酪蛋白為基質，F1和F2受Cu2+、Fe2+、 Zn2+、Mn2+、EDTA和PMSF抑制，以懸浮態幾丁質為基質，F1 和F2分別受（Cu2+、Fe2+、 Zn2+、Mn2+）和（Fe2+、Ca2+）抑制。F2蛋白酶與幾丁質酶活性受SDS抑制；化學合成物對酵素影響，F1和F2蛋白酶分別受到（S10C、S85O）和（S11C、S10C）抑制，F2幾丁質酶受S10C2所抑制；基質特異性之比較：F1和F2對Casein和Azoalbumin皆具有活性以外，F2對Azocasein也具有活性；以酪蛋白為基質，F1和F2的 Km與Vmax為（5 mg/mL、0.24 U/mL）和（5.26mg/mL、0.93 U/mL）；以懸浮態幾丁質為基質，F1和F2的 Km與Vmax為（66.7mg/ mL、5U/L）和（116.7 mg/ mL、30U/L）。 From commercial papain, two proteases ( F1 and F2 ) with chitinase activity were recovered by using DEAE-Sepharose ion-exchange chromatography, CM-Sepharose ion-exchange chromatography, and gel filtration on a Sephacryl S-100 column. When these two enzymes (F1 and F2) were denatured with SDS-PAGE and a reducing agent, F1 and F2 exhibited a single band at 26 kDa and 28 kDa, respectively. When casein and colloidal chitin were used as substrates for measuring the protease and chitinase activities of F1, the optimum pH, pH stability, optimum temperature, and thermal stability were (7, 5-11, 70, 40-60℃) and (4, 5-8, 50℃, 25-50℃) respectively. Measuring with the same substrates, the optimum pH, pH stability, optimum temperature, and thermal stability of F2 were (8, 4-10, 80℃, 25-60℃) and (4, 4-7, 40℃, 30-70℃) respectively. Using casein as the substrate, F1 and F2 were inactived by Cu2+、Fe2+、 Zn2+、Mn2+、EDTA and PMSF . Using colloidal chitin as the substrate, F1 and F2 were inactived by (Cu2+,Fe2+, Zn2+,Mn2+) and (Fe2+,Ca2+). As for effects of chemical substrates on two enzymes, proteases (F1 and F2) were inactived by（S10C, S85O）and（S11C, S10C） while chitinase (F2) was inactived by S10C2. Comparison of the substrate specificity: F1 and F2 act on casein and azoalbumin, besides, F2 also acts on azocasein. With casein as the substrate, the Km and Vmax of F1 and F2 were (5 mg/mL、0.24 U/mL) and (5.26mg/mL、0.93 U/mL) respectively. With colloidal chitin as the substrate, the Km and Vmax of F1 and F2 were (66.7mg/ mL、5U/L) and (116.7 mg/mL、30U/L) respectively.
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