• Journal of Microbiology and Biotechnology
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• v.24 no.1
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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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• v.13 no.1
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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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• v.29 no.2
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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.

• Microbiology and Biotechnology Letters
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• v.43 no.4
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• pp.314-321
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• 2015

Agar-hydrolyzing bacteria were isolated from the coastal sea water of Jeju Island. One isolate, designated as S4, was selected for further study. The S4 cells were Gram-negative and rod-shaped with smooth beige surfaces and single polar flagellum. Cells were grown at $15-42^{\circ}C$, 0.5-5% (w/v) NaCl, between pH 6.0 and 9.0, and in media containing 0.5-5% (w/v) NaCl. The G+C content was 49.93 mol%. The major fatty acids (>15%) were $C_{18:1}{\omega}7c$, $C_{16:0}$ and Summed feature 3 (comprising $C_{16:1}{\omega}7c/iso-C_{15:0}$ 2-OH). Based on 16S rRNA sequencing and biochemical and chemotaxonomic characteristics, the strain was designated as Vibrio sp. S4. In liquid culture supplemented with 0.1% agar the cell density and agarase activity reached a maximum level in 72 h, while agarase activity in the culture without agar was negligible, implying agarose expression is induced by agar. The optimum pH and temperature for the extracellular crude agarase of S4 were 7.0 and $45^{\circ}C$, respectively. However, it also exhibited 98.6% and 87.6% at $40^{\circ}C$ and $50^{\circ}C$, respectively, of the maximum activity seen at $45^{\circ}C$. The crude agarase hydrolyzed agarose into (neo)agarotetraose and (neo)agarohexaose.

• Microbiology and Biotechnology Letters
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• v.44 no.1
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• pp.106-106
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• 2016

• Korean Journal of Microbiology
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• v.52 no.3
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• pp.344-351
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• 2016

A marine bacterium, designated as strain 50C-3, was isolated from a seawater sample collected from the East Sea of South Korea. The strain is a Gram-negative, aerobic, yellow colored polar-flagellated bacterium that grows at $20-50^{\circ}C$ and pH 5.5-8.5. Optimal growth occurred at $40-50^{\circ}C$, at pH 6.5-7.5, and in the presence of 2% (w/v) NaCl. Based on 16S rRNA gene sequence similarity, the isolate was considered to represent a member of the genus Ruegeria. The result of this analysis showed that strain 50C-3 shared 99.4% and 96.98% sequence similarity with Ruegeria intermedia CC-GIMAT-$2^T$ and Ruegeria lacuscaerulensis ITI-$1157^T$, respectively. Furthermore, strain 50C-3 showed clear differences from related strains in terms of several characteristics such as motility, carbon utilization, enzyme production, etc. The DNA G+C content was 66.7 mol%. Chemotaxonomic analysis indicated ubiquinone-10 (Q-10) as the predominant respiratory quinone. Based on phenotypic, chemotaxonomic, and phylogenetic characteristics, the isolate represents a novel variant of the Ruegeria intermedia CC-GIMAT-$2^T$, for which we named Ruegeria sp. 50C-3 (KCTC23890=DSM25519). Strain 50C-3 did not produce cellulase and agarase, but produced alkaline phosphatase, ${\alpha}$-galactosidase, and ${\beta}$-galactosidase. The three enzymes showed stable activities even at $50^{\circ}C$ and thus regarded as thermostable enzymes. Especially, the ${\beta}$-galactosidase activity enhanced by 1.9 times at $50^{\circ}C$ than that at $37^{\circ}C$, which may be very useful for industrial application.