- 作者： 宋賢一; 李偉明; 邱美珠; 張珍田
- 作者服務機構： 國立臺灣大學農業化學系; 私立靜宜女子大學食品營養學系
- 中文摘要：
經由20%丙酮抽取、熱處理、酸沈澱及冷凍乾燥等系列步驟，由洋刀豆純化合乎分析試劑規格之尿素?。尿素?之
萃取係混合1份洋刀豆粉與5份20%丙酮（含1mM EDTA及1mM 2-硫氫化乙醇），於20℃攪拌5分鐘，萃取液之乳
狀物質以熱處理除去之，澄清金黃色萃取液以檸檬酸調整pH值至5.4，沉澱尿素?，經酸沉澱之尿素?溶於0.015 M
磷酸鹽緩衝液，pH 8.5（最終pH 6.8至7.0）中和並冷凍乾燥之。經此等步驟純化，可提高酵素純度14.7倍並獲得63%之活
性收率，1公斤洋刀豆可獲得6.75克之尿素?，其比活性為411單位／毫克蛋白質（240單位／毫克乾物），游離氨含量少
於0.01微克／單位。經由膠體過濾及梯度聚丙烯醯胺膠體電泳顯示此尿素?製品仍含其他蛋白質，尿素?之分子量經
由膠體過濾測得為480,000，然而經由聚丙烯醯胺膠體電泳及活性染色卻顯示兩個尿素?活性帶，其分子量分別為
230,000及480,000。此製品經使用於血液尿素氮（BUN）含量測定結果，顯示此簡易步驟可實際應用於從洋刀豆製備臨
床分析用尿素?。 - 英文摘要： Urease with a purity meeting the requirements of analytical use was purified from jack bean mealthrough steps consisting of 20% acetone extraction, heat treatment, acid precipitation, and lyophilization.For extraction of urease, one part of bean meal was mixed with 5 parts of 20% acetone containing 1 mMEDTA and 1 mM 2-mercaptoethanol, and stirred at 20℃ for 5 min. Milky substances in the extract wereremoved by heat treatment. Urease in the clear yellow supernatant was precipitated by adjusting the pHof the solution to 5.4 with citric acid. The acid precipitated urease was neutralized by dissolving in 0.015 Mphosphate buffer, pH 8.5 (final pH 6·8 to 7·0) and then lyophilized. By this procedure, the purity of theenzyme was increase 14.7 fold, the recovery of activity was 63%, and the yield was 6.75 g from 1 kg ofbean seeds. The specific activity of the preparation was 411 units/mg protein (240 units/mg solid), and thefree ammonia content was less than 0.01 μg per unit. Some other proteins were present in the ureasepreparation as examined by gel filtration and gradient polyacrylamide gel electrophoresis. The molecularweight of the enzyme estimated by gel filtration was 480,000. However, two urease activity bands withmolecular weight of 230,000 and 480,000 were observed in the polyacrylamide gel electrophoregram.From the result of determination of blood urea nitrogen (BUN), this simple purification procedure couldbe used for practical preparation of urease from jack bean meal for clinical analysis.
- 中文關鍵字： urease; jack bean
- 英文關鍵字： --
