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İsolation and biochemical characterization of 8-aminolevulinic acid dehydratase from streptomyces yokosukanensis atcc 25520

By Şengül Aksan

Abstract

Bu çalışmada, nebularin olarak adlandırılan bir pürin ribosit antibiyotiği sentezleyen Streptomyces yokosukanensis ATCC 25520, 5-aminolevulinik asit dehidrataz (ALAD)’mm, amonyum sülfat ile çökeltmesi ve Sefakril S-200’de jel filtrasyon tekniği sonucunda 90.76 kat saflaştırma gerçekleştirilmiştir. İncelenen verilere uygun olarak, enzimin tek polipeptidden oluştuğu ve moleküler ağırlığının 34.8 kDa olduğu SDS-PAGE yöntemi ile saptanmıştır. Enzimin optimum sıcaklığı 45 °C, ve optimum pH ’sı 8 olarak belirlenmiştir. Bazı ağır metallerin; Pb2+ %61, Mg2+ %26, Co2+ %20, Fe3+ %51, Mn2+ %26, Zn2+ %36 +2’ oranlarında inhibe ettiği ve ilginç olarak, Ni nin enzim aktivitesini 15% arttırdığı saptanmıştır. Lineweaver-Burk grafiğinde, Vmax 30.3 |imol PBG/saat/mg protein ve Km değeri 1.21 mmol/reaksiyon karışım bulunmuştur.In this study, S-aminolevulinic acid dehydratase (ALAD) from Streptomyces yokosukanensis ATCC 25520, producer of an unusual purine riboside antibiotic called nebularine, was purified and characterized. Purification procuders involved with ammonium sulphate precipitation and following gel filtration techniques by use of Sephacryl S-200. After Gel filtration a 90.76-fold purification was obtained. According to the data obtained from investigation, the enzyme was found to be a single polypeptide having molecular mass around 34.8 kDa. This was determined by SDS-PAGE. Its optimal temperature around 45 °C, and optimal pH was found to be 8. Some heavy metals inhibited its activity ratio of Pb2+ %61, Mg2+ %26, Co2+ %20, Fe3+ %51, Mn2+ %26, Zn2+ %36. Surprisingly, +2 Ni increased its activity up to 15%. In Lineweaver-Burk plot, Vmax was found as 30.3 |imol PBG/h/mg of protein and Km value 1.21 mmol/reaction mixture

Topics: Alad, Enzim Karakterizasyonu, Streptomycete, Streptomyces Yokosukanensis, Saflaştırma
Year: 2006
OAI identifier: oai:acikerisim.aku.edu.tr:11630/6418
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