Purification and characterization of the extracellular alginate lyase from Streptomyces sp. MET 0515

Abstract

알긴산분해효소(EC 4. 2. 2. 3)를 균체외로 생산하는 새로운 방선균을 전라남도 완도의 연안토양으로부터 분리하고 이 균주를 Streptomyces sp. MET 0515라 명명하였다. 이 균주가 생산하는 균체외 알긴산분해효소를 아세톤 침전, 음이온 교환 크로마토그래피(Q-Sepharose and DEAE-Sepharose), 젤 크로마토그래피(Sephacryl S-200 HR gel filtration chromatography)를 이용하여 정제하였다. 이 효소의 최적 활성 온도과 pH는 각각 <TEX>$70^{\circ}C$</TEX>와 pH 7.5이고, 온도 안정성은 <TEX>$0-50^{\circ}C$</TEX>, pH 안정성은 pH 6.0-9.0였다. 이 효소는 <TEX>$Mn^{2+}$</TEX> 이온 첨가시에는 활성이 증가하였으며 1mM <TEX>$Fe^{3+}$</TEX>, 1mM EDTA, 1mM <TEX>$Zn^{2+}$</TEX> 이온 첨가시에는 활성이 억제되었다. 또한 이 효소는 poly-alpha 1,4-L-guluronate와 poly-beta 1,4-D-mannuronate두 종류의 기질에 대하여 활성을 나타냈다. 따라서 본 효소는 다른 미생물 기원의 효소와 비교해 볼 때 Streptomyces sp.가 생산하는 첫번째 효소로 인정되었다. We isolated a new extracellular alginate lyase-producing microorganism, which displayed alginate-depolymerizing activity in plate assays, from coastal soils in Wando, Jeollanam-do, Korea. This alginate-depolymerizing bacterium belonged to the genus Streptomyces and it was named Streptomyces sp. MET 0515. An extracellular alginate lyase(ALY1) secreted by Streptomyces sp. MET 0515, was purified to homogeneity by a combination of acetone precipitation, anion-exchange chromatography (Q-Sepharose and DEAE-Sepharose) and Sephacryl S-200 HR gel filtration chromatography. Its molecular mass was 26 kDa as determined by SDS-PACE analysis. The enzyme had an optimal temperature of <TEX>$70^{\circ}C$</TEX> for its activity, and was most active at pH 7.5. The thermal and pH stability were <TEX>$0-50^{\circ}C$</TEX>, and pH 6.0-9.0, respectively. The enzyme activity was stimulated by 1mM <TEX>$Mn^{2+}$</TEX>, and inhibited by 1mM <TEX>$Fe^{3+}$</TEX>, 1mM EDTA and 1mM <TEX>$Zn^{2+}$</TEX>. Preliminary analysis of substrate specificity showed that this alginate lyase had activity on both poly-alpha 1,4-L-guluronate and poly-beta 1,4-D-mannuronate in the alginate molecule.