Thermofilik Anoxybacillus sp. AH1'den Üretilen Termostabil β-galaktosidazın Karakterizasyonu

Termofilik bakterilerden elde edilen termostabil β-galaktosidazlar, endüstriyel ve biyoteknolojik uygulamalarda çeşitli avantajlara sahip oldukları için ilgi çekmektedir. Bu çalışmada, Anoxybacillus sp. AH1'den üretilen, oldukça termostabil olan β-galaktosidaz, saflaştırıldı ve karakterize edildi. En yüksek enzim üretimi, bakterinin 24 saat inkübe edilmesinden sonra elde edildi. Enzim, amonyum sülfat çöktürmesi, diyaliz ve jel filtrasyon kromatografisi (Sephadex G-75) kullanılırak saflaştırıldı. Saflaştırma aşamalarından sonra, β-galaktosidazın % 13,9 verimle 10,2 kata kadar saflaştırıldığı tespit edildi. β-galaktosidazın moleküler kütlesi, SDS-PAGE ile 75 kDa olarak tahmin edildi. Saflaştırılmış enzimin oldukça stabil olduğu ve 120 dakika sonunda 60 ° C'de orijinal aktivitenin% 71'ini, 70 ° C'de ise % 53'ünü koruduğu tespit edildi. Saflaştırılmış β-galaktosidazın Km ve Vmax değerleri sırasıyla 1,249 mM ve 0,5 μmol dakika-1 olarak hesaplandı. Ca2+, Zn2+ ve Mg2+ β-galaktosidaz aktivitesini önemli ölçüde aktive ederken, Cu2+ ve metal iyon şelatörleri, 1,10-phenanthroline (phen) ve ethylenediaminetetraacetic acid (EDTA) enzim aktivitesini önemli ölçüde inhibe etmiştir. Saflaştırılmış β-galaktosidaz aktivitesi 2 mM PMSF (phenylmethylsulfonyl fluoride), PCMB (p-chloromercuribenzoic acid), DTT (dithiothreitol), ve β-ME (β-mercaptoethanol) ile artar iken, 1 mM NEM (N-ethylmaleimide) ile tamamen inhibe edildiği belirlendi.

Anahtar Kelimeler:

enzim aktivitesi inhibisyonu, termostabil b-galaltosidaz, enzim saflaştırma, Anoxybacillus sp. AH1.

Characterization of a Thermally Stable β-galactosidase Produced by Thermophilic Anoxybacillus sp. AH1

Thermostable β-galactosidases from thermophilic bacteria have attracted increasing interest to have various advantages in industrial and biotechnological applications. In this study, a highly thermally stable β-galactosidase produced by Anoxybacillus sp. AH1was purified and characterized. The highest enzyme production was achieved after the bacterium was incubated for 24 hours. The enzyme was purified by precipitation with ammonium sulphate dialysis, gel filtration chromatography using Sephadex G-75. After the purification steps, β-galactosidase was found to be purified 10.2-fold and a yield of 13.9%. The molecular mass of the galactosidase was estimated to be 75 kDa by SDS-PAGE. The purified enzyme was highly stable and retained at 71% of the original activity at 60 °C and 53% at 70 oC within 120 minutes. The Km and Vmax values of purified β-galactosidase were calculated as 1.249 mM and 0.5 μmol minutes-1, respectively. Ca2+, Zn2+, and Mg2+ significantly activated β-galactosidase activity, whereas enzyme activity was inhibited significantly by Cu+2 as well as by the metal ion chelators1,10-phenanthroline (phen) and ethylenediaminetetraacetic acid (EDTA). The Purified β-galactosidase activity was increased by PMSF (phenylmethylsulfonyl fluoride), PCMB (p-chloromercuribenzoic acid), DTT (dithiothreitol), and β-ME (β-mercaptoethanol) at 2 mM, but inhibited completely by NEM (N-ethylmaleimide) at 1 mM.

Keywords:

enzyme activity inhibition, thermostable B-galactosidase, enzyme purification, Anoxybacillus sp. AH1,

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