Stano J, Mićieta K, Neubert K, Luckner M, Adam H
Faculty of Pharmacy, Department of Botany, Faculty of Sciences, Comenius University, Bratislava, Slovak Republic.
Pharmazie. 2002 Mar;57(3):176-7.
A simple, rapid and reproducible procedure for the identification of extracellular Californian poppy (Eschscholzia californica Cham.) beta-galactosidase is described using callus cultures of seedlings from the tested plant, roots of 4-days-old seedlings of Californian poppy germinating on agar plates and cell suspension cultures cultivated from callus cultures. 6-Bromo-2-naphthyl-beta-D-galactopyranoside and p-nitrophenyl-beta-D-galactopyranoside were used as substrates for the determination of the intracellular and extracellular activities of beta-galactosidase. The extracellular beta-galactosidase activity was identified by evaluating the dye-zones in an agar medium. The enzyme from Californian poppy callus cultures or from seedling roots cultivated on agar plates supplemented with 6-bromo-2-naphthyl-galactopyranoside hydrolyzed this substrate releasing 6-bromo-2-naphthol. By simultaneous coupling with hexazonium p-rosaniline the corresponding (reddish-brown) azo-dye was formed. The agar plate method described permits rapid, simple and specific detection of plant producers of extracellular beta-galactosidase.
本文描述了一种简单、快速且可重复的方法，用于鉴定加州罂粟（Eschscholzia californica Cham.）的细胞外β-半乳糖苷酶。该方法使用受试植物幼苗的愈伤组织培养物、在琼脂平板上萌发4天的加州罂粟幼苗的根以及从愈伤组织培养物中培养的细胞悬浮培养物。6-溴-2-萘基-β-D-吡喃半乳糖苷和对硝基苯基-β-D-吡喃半乳糖苷用作测定β-半乳糖苷酶细胞内和细胞外活性的底物。通过评估琼脂培养基中的染色带鉴定细胞外β-半乳糖苷酶活性。来自加州罂粟愈伤组织培养物或在补充有6-溴-2-萘基半乳糖苷的琼脂平板上培养的幼苗根中的酶水解该底物，释放出6-溴-2-萘酚。通过与对品红六偶氮盐同时偶联，形成相应的（红棕色）偶氮染料。所描述的琼脂平板法允许快速、简单且特异性地检测细胞外β-半乳糖苷酶的植物产生者。
