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題 名 | 四季豆果膠酯酶之純化與其特性之研究 |
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作 者 | 陳家伯; 區少梅; | 書刊名 | 食品科學 |
卷 期 | 21:2 1994.04[民83.04] |
頁 次 | 頁83-95 |
分類號 | 463 |
關鍵詞 | 果膠酯酶; 純化; 特性; 四季豆; Pectinesterase; Purification; Characterization; Green bean; |
語 文 | 中文(Chinese) |
中文摘要 | 四季豆果膠酯?之抽取以四季豆重量及1.5M氯化鈉溶液體積比例為1:3時,可得較佳之抽取率,此粗酵素液經50~90%飽和硫酸銨沉澱分離,回收約70%的酵素活性,並提高比活性2.4倍,再經過陰離子交換層析加上膠體過濾層析,能夠有效純化果膠酯?,並分離出兩種分子型態PE-I與PE-II,回收率及純化倍數分別為10.64%,502倍及22.19%,324倍。經純化所得之PE-I與PE-Ⅱ分子量各為45,000及55,000 daltons。 PE-I之最適作用溫度及pH值為70℃及pH7,而PE-II為60℃及pH6-7,兩者均在pH8環境下有最佳之穩定性。PE-1作用於果膠質(DE49%)之Vmax及Km為1.63μeq COOH/min及0.71g/L,而PE-II之Vmax及Km為3.41μeq COOH/min及2.02g/L。聚半乳糖醛酸對四季豆果膠酯?呈現競爭性抑制作用,對PE-I其抑制常數為2.09g/L,對PE-II為2.02g/L。金屬離子的存在可大幅提升PE-I及PE-II的活性,但高濃度的二價金屬離子容易使果膠質溶液產生凝膠,降低酵素及基質分子之移動性而減低PE-I及PE-II反應速率。另外,少量N-Bromosuccinimide之添加可使果膠酯?之PE-I及PE-II完全喪失活性,因此了解鹼性胺基酸在其活性上佔有重要之地位。 |
英文摘要 | It was found appropriate and convenient to extract pectinesterase from green beans with 1.5M NaC1 solution. The crude pectinesterase extract was subjected to ammonium sulfate fractionation and about 70~ of the enzyme activity was recovered with purification of about 2.4 folds by collecting the precipitate of 50~90% saturation of ammonium sulfate. Application of DEAE Sepharose chromatography and gel filtration chromatography after ammonium sulfate fractonation, enabled efficient purification of pectinesterase from green beans, as well as the separation of two forms of the enzyme, PE-I and PE-II. PE-I was purified about 502 folds with an activity recovery of 10.64%, while PE-II was purified about 324 folds with an activity recovery of 22.19%. The molecular weights of PE-I and PE-II were 45,000 and 55,000 dalton, respectively. The optimum temperature for the activities of PE-I and PE-II were 70℃ and 60℃, respectively. The optimum pH of PE-I for the reaction was pH 7 while for PE-II was pH 6-7. The pH stability of the two isozymes was both at pH8.0. The Km values of PE-I and PE-II were 0.71 and 2.02 g/L (pectin DE 49%), and the Vmax values were 1.63 and 3.41μeq COOH/min, respectively. Polygalacturonic acid exhibited competitive inhibition upon the PE-I and PE-II, with K1 values of 2.09 and 2.02g/L, respectively. The presence of salts in the media could increase PE-I and PE-II activity, but high concentrations of divalent metal ions decreased PE-I and PE-II activity. Because the high concentrations of divalent metal ions caused gel formation of pectin solution, the mobility of PE-I and PE-II and substrate molecules decreased and the rate of PE-I and PE-II reaction slowed down. The N-Bormosuccinimide modification of PE-I and PE4I resulted in specific inactivation. Therefore, the basic amino acids should be involved in the activity of pectinesterase. |
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