• Journal of Microbiology and Biotechnology
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• 제24권1호
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• pp.19-25
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• 2014

An extracellular agarase was purified from Bacillus sp. BI-3, a thermophilic agar-degrading bacterium isolated from a hot spring in Indonesia. The purified agarase revealed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular mass of 58 kDa. The optimum pH and temperature of the agarase were 6.4 and $70^{\circ}C$, respectively. The activity of the agarase was stable at high temperatures, and more than 50% activity was retained at $80^{\circ}C$ for 15 min. Furthermore, the enzyme was stable in the pH range of 5.8-8.0, and more than 60% of the residual activity was retained. Significant activation of the agarase was observed in the presence of $K^+$, $Na^+$, $Ca^{2+}$, $Mg^{2+}$, and $Sr^{2+}$; on the other hand, $Ba^{2+}$, $Zn^{2+}$, $Cu^{2+}$, $Mn^{2+}$, $Co^{2+}$, $Fe^{2+}$, and EDTA inhibited or inactivated the enzyme activity. The components of the hydrolytic product analyzed by thin-layer chromatography showed that the agarase mainly produced neoagarobiose. This study is the first to present evidence of agarolytic activity in aerobic thermophilic bacteria.

• Fisheries and Aquatic Sciences
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• 제13권1호
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• pp.36-48
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• 2010

An agar-degrading Agarivorans sp. AG17 strain was isolated from the red seaweed Grateloupia filicina collected from Jeju Island. A beta-agarase gene from Agarivorans sp. AG17 was cloned and designated as agrA. agrA has a 2,985 bp coding region encoding 995 amino acids and was classified into the glycoside hydrolase family (GHF)-50. Predicted molecular mass of the mature protein was 105 kDa. His-tagged agrA was overexpressed in Escherichia coli and purified as a fusion protein. The enzyme showed 158.8 unit/mg specific activity (optimum temperature at $65^{\circ}C$ and pH 5.5 in acetate buffer) with unique biochemical properties (high thermal and pH stabilities). Enzyme produced neoagarohexaose, neoagarotetraose and neoagarobiose by degrading agar, and hydrolyzed neoagaro-oligosaccharides were biologically active. Hence the purified enzyme has potential for use in industrial applications such as the development of cosmetics and pharmaceuticals.

• Journal of Microbiology and Biotechnology
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• 제29권2호
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• pp.235-243
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• 2019

A special eosinophilic agarase exo-type ${\beta}$-agarase gene, AgaDL6, was cloned from a marine agar-degrading bacterium, Flammeovirga sp. HQM9. The gene comprised 1,383-bp nucleotides encoding a putative agarase AgaDL6 of 461 amino acids with a calculated molecular mass of 52.8 kDa. Sequence analysis revealed a ${\beta}$-agarase domain that belongs to the glycoside hydrolase family (GH) 16 and a carbohydrate-binding module (CBM_4_9) unique to agarases. AgaDL6 was heterologously expressed in Escherichia coli BL21 (DE3). Enzyme activity analysis of the purified protein showed that the optimal temperature and pH of AgaDL6 were $50^{\circ}C$ and 3.0, respectively. AgaDL6 showed thermal stability by retaining more than 98% of activity after incubation for 2 h at $50^{\circ}C$, a feature quite different from other agarases. AgaDL6 also exhibited outstanding acid stability, retaining 100% of activity after incubation for 24 h at pH 2.0 to 5.0, a property distinct from other agarases. This is the first agarase characterized to have such high acid stability. In addition, we observed no obvious stimulation or inhibition of AgaDL6 in the presence of various metal ions and denaturants. AgaDL6 is an exo-type ${\beta}$-1,4 agarase that cleaved agarose into neoagarotetraose and neoagarohexaose as the final products. These characteristics make AgaDL6 a potentially valuable enzyme in the cosmetic, food, and pharmaceutical industries.

• 한국미생물·생명공학회지
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• 제43권4호
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• pp.314-321
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• 2015

대한민국 제주도 연안 해수로부터 agarase를 생산하는 균주 S4를 분리하였다. 균주 S4는 그람-음성의 막대형 세포로 부드러운 베이지색 원형 콜로니를 형성하며, 한 개의 극성 편모를 갖는다. S4 균주는 $15-42^{\circ}C$, 0.5-5%(w/v) NaCl, pH 6.0-9.0, 0.5-5%(w/v) NaCl 농도에서 안정된 성장을 보인다. S4 균주의 G+C content는 49.93 mol%, 세포내 주요 지방산 (>15%)은 $C_{18:1}{\omega}7c$, $C_{16:0}$, Summed feature 3(comprising $C_{16:1}{\omega}7c/iso-C_{15:0}$ 2-OH)이다. 16S rRNA 염기서열, 생화학적 및 분류학정 특징에 기초하여 S4 균주를 Vibrio sp. S4로 명명하였다. 0.1% agar를 첨가한 액체배지에서 S4 균주는 72시간에 세포농도와 agarase 활성이 최대치를 보였다. 반면, agar 를 첨가하지 않은 배양액에서의 agarase 활성은 무시할만한 수준이었으며, 이는 균주의 agarase 유전자 발현이 agar에 의해 유도됨을 시사하고 있다. 균주 S4가 세포외부로 분비하는 총 agarase는 $45^{\circ}C$와 pH 7.0에서 최상의 효소 활성을 보였으며, agarose를 분해하여 (neo)agarotetraose와 (neo)agarohexaose를 생산하였다.

• 한국미생물·생명공학회지
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• 제44권1호
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• pp.106-106
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• 2016

• 미생물학회지
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• 제52권3호
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• pp.344-351
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• 2016

대한민국 동해안 울진 앞 바닷물로부터 50-C로 명명한 해양 미생물을 분리하였다. 50-C 균주는 그람-음성, 호기성 세균이며, 노란색 집락을 형성하고, 극성편모를 갖는 박테리아이다. 이 균주는 $20-50^{\circ}C$, pH 5.5-8.5 범위에서 자라며, 비교적 고온인 $40-50^{\circ}C$, pH 6.5-7.5, 2% (w/v) NaCl에서 최적 성장을 보인다. 16S rRNA 유전자 서열 분석결과 50C-3 균주는 Ruegeria 속에 속하는 R. intermedia CC-GIMAT-$2^T$, R. lacuscaerulensis ITI-$1157^T$의 16S rRNA 유전자 서열과 각각 99.4%, 96.98% 상동성을 보였다. 그러나 50C-3 균주는 운동성, 탄소이용능력, 효소생산능력 등의 생리학적 특성에서 두 균주와는 명확히 다른 특성을 보였다. 50C-3 균주의 DNA G+C content는 66.7 mol%이고, 주요한 respiratory quinone은 ubiquinone-10 (Q-10)이었다. 이와 같은 형태학적, 생리학적, 유전학적 특성을 비교하여, 50C-3 균주는 R. intermedia CC-GIMAT-$2^T$와 같은 종에 속하는 새로운 변종으로 판단되며 Ruegeria sp. 50C-3으로 명명하였다(KCTC23890 =DSM25519). 50C-3 균주는 cellulase, agarase 활성은 없었지만, alkaline phosphatase, ${\alpha}$-galactosidase, ${\beta}$-galactosidase를 생산하였고 이들 모두 $50^{\circ}C$ 에서도 활성이 좋은 내열성 효소일 것으로 판단되었다. 특히, ${\beta}$-galactosidase의 경우 $37^{\circ}C$에서 보다 $50^{\circ}C$에서의 활성이 1.9배 증가하여 산업적으로 활용성이 클 것으로 예상된다.