Isolation of an Agarase-producing Persicobacter sp. DH-3 and Characterization of its β-agarase

Agarase를 생산하는 Persicobacter sp. DH-3의 분리 및 β-agarase의 특성

Abstract

The purpose of this study was to isolate a new marine agarase-producing bacterium. Agarase can hydrolyze agar and agarose to produce agarooligosaccharides or neoagarooligosaccharides, which possess many physiological functions. Strain DH-3 was isolated from seawater collected from the coast of Yeosu at Jeollanam province, Korea. A 16S rDNA sequence analysis showed this strain to be Persicobacter sp. DH-3. Extracellular agarase was prepared from culture media of Persicobacter sp. DH-3 and used for characterization. Relative activities at 20, 30, 40, 50, 60, and $70^{\circ}C$ were 50, 55, 70, 100, 90, and 50%, respectively. Relative activities at pH 5, 6, 7, and 8 were 75, 100, 90, and 75%, respectively. The enzyme showed maximum activity at $50^{\circ}C$ in a 20 mM Tris-HCl buffer at pH 6. This enzyme could be useful, as agar is in liquid state at $50^{\circ}C$. Agarase activities were maintained at 80% or more for 2 hr at 20, 30, and $40^{\circ}C$. Thin layer chromatography analysis suggested that Persicobacter sp. DH-3 produced extracellular ${\beta}$-agarases as it hydrolyzed agarose to produce neoagarohexaose and neoagarotetraose. In addition, zymogram analysis confirmed that Persicobacter sp. DH-3 produces at least three agar-degrading enzymes with molecular weights of 45, 70, and 140 kDa. Therefore, it is expected that agarases from Persicobacter sp. DH-3 could be used to produce functional neoagarooligosaccharides.

이 연구의 목적은 agarase를 생산하는 새로운 해양 박테리아를 분리하는 것이다. Agarase는 agarose를 가수분해하여 다양한 생리활성을 가지는 agarooligosaccharides 또는 neoagarooligosaccharides를 생성할 수 있다. 전라남도의 여수연안에서 채취한 해수로부터 DH-3 균주를 분리하였다. DH-3 균주는 16S rDNA 서열분석을 통하여 Persicobacter sp. DH-3로 명명하였다. 세포 외로 분비된 효소는 Persicobacter sp. DH-3의 배양액으로부터 얻었으며, 특성연구에 사용하였다. 20, 30, 40, 50, 60 및 $70^{\circ}C$에서의 온도별 상대활성은 각각 50, 55, 70, 100, 90 및 50%였다. pH 5, 6, 7 및 8에서의 pH별 상대활성은 각각 75, 100, 90, 75%였다. 본 효소는 20 mM Tris-HCl 완충액에서 pH 6.0 및 $50^{\circ}C$에서 최대활성을 나타내었다. 본 효소는 한천이 액체상태로 존재하는 $50^{\circ}C$에서 최대활성을 나타내었으므로 산업적으로 유용한 효소로 판단된다. 본 효소는 20, 30 및 $40^{\circ}C$에서 2시간 동안 열처리하여도 80% 이상의 잔존활성을 보였다. TLC 분석결과, Persicobacter sp. DH-3 균주의 한천분해효소는 네오한천올리고당인 neoagarohexaose와 neoagarotetraose를 생성하였으므로 ${\beta}$-agarase로 확인되었다. Zymogram 분석결과, Persicobacter sp. DH-3는 적어도 45, 70 및 140 kDa 크기의 3종류 이상의 한천분해효소를 생산하는 것을 확인할 수 있었다. 그러므로, Persicobacter sp. DH-3 유래 agarase는 기능성을 가진 neoagarooligosaccharides의 생산에 활용될 수 있을 것으로 기대된다.

Fig. 1. Phylogenetic tree based on almost complete 16S rDNA sequence comparing strain DH-3 with other bacteria. The numbers at the branch are bootstrap values and numbers in parenthesis are accession numbers at NCBI.

Fig. 2. The growth curve (□) of Persicobacter sp. DH-3 and the production of agarase (■) in the presence of 0.2% (w/v) agar. The highest level of agarase was reached at the period of lag phase.

Fig. 3. Effects of reaction temperatures on agarase activities. The reactions were carried out at 20, 30, 40, 50, 60 and 70℃ in 1 ml of 20 mM Tris-HCl (pH 6.0) buffer and 0.5 ml of enzyme solution for 30 min.

Fig. 4. Effects of pHs on the agarase activities. 20 mM Tris-HCl buffer (pH 5.0-8.0) and GTA buffer (pH 8.0-9.0) were used. The reactions were carried out at 50℃ in 1 ml of corresponding buffer and 0.5 ml of enzyme solution for 30 min.

Fig. 5. Remaining activities of agarase after heat treatments for defined times. The enzyme solutions were pre-incubated at 20 (■), 30 (◆), 40 (▼), 50 (●), 60 (○) and 70℃ (□) for 0, 0.5, 1.0, 1.5, 2.0 hr. The reactions were then carried out at 50℃ in 1 ml of 20 mM Tris-HCl (pH 6.0) buffer containing 0.5 ml of heat-treated enzyme solution for 30 min.

Fig. 6. Zymogram analysis of agarases. The molecular masses of the enzymes were 45, 70 and 140 kDa. (Lane M size markers; lane E, agarases).

Fig. 7. TLC analysis of the hydrolyzed products of agarose by agarase. The reactions were carried out at 50℃ in Tris-HCl (pH 6.0) buffer containing 0.2% (w/v) agar and 0.5 ml of enzyme solution for 5, 10, 15, 20, 25 and 30 min. The reaction mixtures were developed by TLC. (G, Dgalactose; NA, neoagarooligosaccharides; NA2, neoagarobiose; NA4, neoagarotetraose; NA6, neoagarohexaose).