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

張雅敏(Ya-Min Chang) ； 鍾雲琴(Yun-Chin Chung) ； 徐家蓁(Chia-Chen Hsu) ； 陳麗純(Li-Chun Chen) ； 江翠蓮(Chui-Liang Chiang) ； 張珍田(Chen-Tien Chang) ； 宋賢一(Hsien-Yi Sung)

《Botanical Studies》 52卷1期 (2011/01) Pp. 23-34

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-D-glucosaminide (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℃，K(下標 m)值為1.3 mM而V(下標 max)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℃，保溫60分鐘幾無活性損失，頗為穩定，但55℃以上則顯著失去活性，重金屬離子Hg(上標 2+)(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pNP-β-GlcNAc和pNP-β-GalNAc二基質係屬同一活性中心。

關鍵字

無花果Ficus carica Linn.乳汁 ； β-N-乙醯胺基六碳糖苷酶 ； 純化 ； 性質

並列摘要

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℃, the Km was 1.3 mM, the V(subscript max) was 6.0 μmol mi^(n-1) mg^(-1) and the activation energy was 8.93 kcal/mol. The molecular mass of the enzyme was 13.7kDa, 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℃, but its activity decreased significantly at temperatures greater than 55℃. Both the heavy metal ion Hg(superscript 2+) (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.

並列關鍵字

Characterization ； Fig Ficus carica latex ； β-N-acetylhexosaminidase ； Purification