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題名 | Biochemical Characterization of a β-N-Acetylhexosaminidase from Fig Latex=無花果乳汁 β-N-乙醯胺基六碳糖苷酶之生化性質研究 |
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作者姓名(中文) | 張雅敏; 鍾雲琴; 徐家蓁; 陳麗純; 江翠蓮; 張珍田; 宋賢一; |
作者姓名(外文) | Chang, Ya-min; Chung, Yun-chin; Hsu, Chia-chen; Chen, Li-chun; Chiang, Chui-liang; Cgang, Chen-tien; Sung, Hsien-yi; |
書刊名 | Botanical Studies |
卷期 | 52:1 2011.01[民100.01] |
頁次 | 頁23-34 |
分類號 | 435.337 |
語文 | eng |
關鍵詞 | 無花果乳汁; β-N-乙醯胺基六碳糖苷酶; 純化; 性質; Characterization; Fig latex; Ficus carica latex; β-N-acetylhexosaminidase; Purification; Ficus carica linn; |
中文摘要 | 無花果 (Ficus carica Linn.) 乳汁所含 β-N-乙醯胺基六碳糖苷酶 (β-NAHA)經由 pAPMA-Sepharose CL-4B管柱親和性吸附去除硫醇型蛋白酶、 Sephacryl S-100 HR膠體過濾層析和 DEAE-Sephacel離子交換層析等三管柱層析步驟純化,可得一純化之主要 β-NAHA,以 SDS-PAGE及 β-NAHA活性染色分析顯示純化之 β-NAHA幾乎已達均質純度。純化之 β-NAHA具水解 p-nitrophenyl-N-acetyl-β-Dglucosaminide (pNP-β-GlcNAc) 及 p-nitrophenyl-N-acetyl-β-D-galactosaminide (pNP-β-GalNAc)活性,其水解 pNP-β-GlcNAc之最適 pH為 4.5,最適溫度為 60°C,Km值為 1.3 mM而 Vmax為 6.0 μmol min-1 mg-1。以膠體過濾法測得酵素分子量為 13.7 kDa,以等電焦集電泳及活性染色測得酵素等電點為 pH 3.5,熱穩定性分析顯示酵素於 30至 50°C,保溫 60分鐘幾無活性損失,頗為穩定,但 55°C以上則顯著失去活性,重金屬離子 Hg2+ (0.25 mM) 及化學修飾劑 diethyl pyrocarbonate (2.5 mM) 顯著抑制酵素活性,以基質專一性及混合基質競爭動力學分析顯示純化之 β-NAHA對 β組態醣苷鍵具專一性,而催化水解 pNPβ-GlcNAc和 pNP-β-GalNAc二基質係屬同一活性中心。 |
英文摘要 | A major isoform of β-N-acetylhexosaminidase (β-NAHA) (EC 3.2.1.52) was purified from fig latex in three chromatography steps, affinity chromatography on pAPMA-Sepharose CL-4B to remove cysteine proteases, Sephacryl S-100 HR gel filtration, and DEAE-Sephacel ion-exchange chromatography. The purified β-NAHA appeared almost homogeneous on SDS-polyacrylamide gel electrophoresis and enzyme-activity staining. The purified enzyme catalyzed the hydrolysis of both p-nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-β-GlcNAc) and p-nitrophenyl-N-acetyl-β-D-galactosaminide (pNP-β-GalNAc). The optimum pH for the pNP-β-GlcNAc hydrolysis was 4.5, the optimum temperature was 60°C, the Km was 1.3 mM, the Vmax was 6.0 µmol min-1 mg-1 and the activation energy was 8.93 kcal/mol. The molecular mass of the enzyme was 13.7 kDa, as estimated by gel filtration. The isoelectric point of the enzyme was 3.5, as estimated by isoelectric focusing electrophoresis and activity staining. The enzyme was thermally stable after 60 min at 30-50°C, but its activity decreased significantly at temperatures greater than 55°C. Both the heavy metal ion Hg2+ (0.25 mM) and the chemical modification reagent diethyl pyrocarbonate (2.5 mM) significantly inhibited enzyme activity. Substrate specificity and competition kinetics analysis indicated that the activity of the purified β-NAHA was specific for the β-glycosidic linkage, and the enzyme had only one active site for substrates, pNP-β-GlcNAc and pNP-β-GalNAc. |
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