Screening for Natural Inhibitors of Penicillinase by Copolymerization of Hydrolyzed Starch or Glycogen in Sodium Dodecylsulfate Polyacrylamide Gels for Detecting Penicillinase Activity

以水解過之澱粉或肝醣共聚合於SDS-聚丙烯酸胺膠體中检测盤尼西林水解酶活性作為盤尼西林水解酶天然抑制物之篩選

梁文俐(Wen-Li Liang) ； 黄惠曼(Hui-Man Huang) ； 林榮第(Rong-Dih Lin) ； 侯文琪(Wen-Chi Hou) ； 梁文俐(Wen-Li Liang) ； 黄惠曼(Hui-Man Huang) ； 林榮第(Rong-Dih Lin) ； 侯文琪(Wen-Chi Hou)

《Botanical Bulletin of Academia Sinica》 44卷3期 (2003/07) Pp. 187-191

https://doi.org/10.7016/BBAS.200307.0187

摘要

以0.08%水解過濺粉或肝醣共聚合於7.5%或10%SDS－聚丙烯醯胺膠體中。經遇電泳與移除 SDS 的步驟俊，在膠體中的磐尼西林水解酶和盤尼西林G(100mg在50mL, 0.1M磷酸緩衡液，pH7.0)反應30鐘俊，以0.6% I2在6%KI溶液中染色。盤尼西林水解酶分别在紫色(水解遇溅粉)或是橘红色(肝酪)的背景色下，呈现反白的活性带。利用此活性染色法，检测商品化盤尼西林水解酶(来自Bacillus cereus)以及自行培养的 methicillin－抗性的Stahylococcus aureus ATCC33591株。利用此活性染色法運用放盤尼西林水解酶天然抑制物之篩選。结果提示,發现葱醒颊(anthraquinone-related)化合物，例如 aloe-emodin, emodin and rhein，都能抑制盤尼西林水解酶的活性。此－快速且靈敏的方法可以莲用放蟹尼西林水解酶的钝化、特性分析及天然抑制物的篩選上。

關鍵字

活性染色 ； 共聚合 ； 盤尼西林水解酶 ； 蔥醌類

並列摘要

The 0.08% hydrolyzed starch or glycogen were copolymerized in 7.5% or 10% sodium dodecylsulfate polyacrylamide gels. After electrophoresis and SDS removal, the commercial penicillinase in gels was reacted with penicillin G (100mg in 50mL, 0.1 M phosphate buffer, pH 7.0) for 30 mm and then stained with 0.6% 12 in 6% KI solutions. The clear zone of penicillin　are activity bands stood out against purple or orange-red backgrounds, respectively, for hydrolyzed starch or glycogen used. This activity staining method was used successfully to detect commercial penicillinase activities from Bacillus cereus and the cultured methicillin-resistant Staphylococcus aureus ATCC 33591 strain. This activity staining method was also applied to penicillinase natural inhibitor screenings. It was found that anthraquinone-related compounds, such as aloe-emodin, emodin and rhein, could inhibit penicillinase activity. This fast and sensitive method can be used in the process of penicillinase purification, characterization and inhibitor screening.

並列關鍵字

Activity staining ； Anthraquinone ； Copolymerization ； Penicillinase ； SDS-PAGE