• Fisheries and Aquatic Sciences
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• v.11 no.2
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• pp.76-81
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• 2008

We isolated 1,916 strains of bacteria from gut and feces of abalone. The most active agarolytic bacterium, DAG-130, was identified from the gut of the abalone Haliotis gigantea. Of the bacteria harbored by both H. discus hannai and H. gigantea, 59% were agarolytic. There was no significant difference in the number of agarolytic bacteria isolated from abalone fed on the seaweeds Gelidium amansii, Laminaria japonica, or Undaria pinnatifida. Of the agarolytic bacteria, 72% were isolated from the guts of all sources tested while 43% came from the feces. The strain DAG-130 showed 100% identity with the bacterium Vibrio cyclotrophicus based on phylogenetic analysis of l6S rDNA. The bacterium produced monomers and oligomers from the agar substrate.

• Journal of Microbiology and Biotechnology
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• v.20 no.10
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• pp.1378-1385
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• 2010

A novel agarolytic bacterium, KY-YJ-3, producing extracellular agarase, was isolated from the freshwater sediment of the Sincheon River in Daegu, Korea. On the basis of Gram-staining data, morphology, and phylogenetic analysis of the 16S rDNA sequence, the isolate was identified as Cellvibrio sp. By ammonium sulfate precipitation followed by Toyopearl QAE-550C, Toyopearl HW-55F, and MonoQ column chromatographies, the extracellular agarase in the culture fluid could be purified 120.2-fold with a yield of 8.1%. The specific activity of the purified agarase was 84.2 U/mg. The molecular mass of the purified agarase was 70 kDa as determined by dodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal temperature and pH of the purified agarase were $35^{\circ}C$ and pH 7.0, respectively. The purified agarase failed to hydrolyze the other polysaccharide substrates, including carboxymethyl-cellulose, dextran, soluble starch, pectin, and polygalacturonic acid. Kinetic analysis of the agarose hydrolysis catalyzed by the purified agarase using thin-layer chromatography showed that the main products were neoagarobiose, neoagarotetraose, and neoagarohexaose. These results demonstrated that the newly isolated freshwater agarolytic bacterium KY-YJ-3 was a Cellvibrio sp., and could produce an extracellular ${\beta}$-agarase, which hydrolyzed agarose to yield neoagarobiose, neoagarotetraose, and neoagarohexaose as the main products.

• Journal of Microbiology and Biotechnology
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• v.22 no.9
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• pp.1237-1244
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• 2012

Agarase genes of non-marine agarolytic bacterium Cellvibrio sp. were cloned into Escherichia coli and one of the genes obtained using HindIII was sequenced. From nucleotide and putative amino acid sequences (713 aa, molecular mass; 78,771 Da) of the gene, designated as agarase AgaA, the gene was found to have closest homology to the Saccharophagus degradans (formerly, Microbulbifer degradans) 2-40 aga86 gene, belonging to glycoside hydrolase family 86 (GH86). The putative protein appears to be a non-secreted protein because of the absence of a signal sequence. The recombinant protein was purified with anion exchange and gel filtration columns after ammonium sulfate precipitation and the molecular mass (79 kDa) determined by SDS-PAGE and subsequent enzymography agreed with the estimated value, suggesting that the enzyme is monomeric. The optimal pH and temperature for enzymatic hydrolysis of agarose were 6.5 and $42.5^{\circ}C$, and the enzyme was stable under $40^{\circ}C$. LC-MS and NMR analyses revealed production of a neoagarobiose and a neoagarotetraose with a small amount of a neoagarohexaose during hydrolysis of agarose, indicating that the enzyme is a ${\beta}$-agarase.

• Journal of Microbiology and Biotechnology
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• v.31 no.5
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• pp.756-763
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• 2021

Agarose is a linear polysaccharide composed of ᴅ-galactose and 3,6-anhydro-ʟ-galactose (AHG). It is a major component of the red algal cell wall and is gaining attention as an abundant marine biomass. However, the inability to ferment AHG is considered an obstacle in the large-scale use of agarose and could be addressed by understanding AHG catabolism in agarolytic microorganisms. Since AHG catabolism was uniquely confirmed in Vibrio sp. EJY3, a gram-negative marine bacterial species, we investigated AHG metabolism in Streptomyces coelicolor A3(2), an agarolytic gram-positive soil bacterium. Based on genomic data, the SCO3486 protein (492 amino acids) and the SCO3480 protein (361 amino acids) of S. coelicolor A3(2) showed identity with H2IFE7.1 (40% identity) encoding AHG dehydrogenase and H2IFX0.1 (42% identity) encoding 3,6-anhydro-ʟ-galactonate cycloisomerase, respectively, which are involved in the initial catabolism of AHG in Vibrio sp. EJY3. Thin layer chromatography and mass spectrometry of the bioconversion products catalyzed by recombinant SCO3486 and SCO3480 proteins, revealed that SCO3486 is an AHG dehydrogenase that oxidizes AHG to 3,6-anhydro-ʟ-galactonate, and SCO3480 is a 3,6-anhydro-ʟ-galactonate cycloisomerase that converts 3,6-anhydro-ʟ-galactonate to 2-keto-3-deoxygalactonate. SCO3486 showed maximum activity at pH 6.0 at 50℃, increased activity in the presence of iron ions, and activity against various aldehyde substrates, which is quite distinct from AHG-specific H2IFE7.1 in Vibrio sp. EJY3. Therefore, the catabolic pathway of AHG seems to be similar in most agar-degrading microorganisms, but the enzymes involved appear to be very diverse.

• Journal of Life Science
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• v.11 no.4
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• pp.291-297
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• 2001

The marine bacterium isolated from Porphyra dentata showing green spot rot disease was identified as Pseudomonas sp. the strain have CNCase activity, xylanase activity and protease activity as well as agarase activity. But the strain has no pectate lyase activity. Porphyra dentata tissue inoculated this isolate was macerated after 1 week incubation. The characteristics of extracellular crude agarase of this isolate were examined, the optimal pH and temperature were pH7 and 3$0^{\circ}C$, respectively.

• 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.

• Microbiology and Biotechnology Letters
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• v.43 no.2
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• pp.134-141
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• 2015

An agarolytic marine bacterium (H9) was isolated from the coastal seawater of the West Sea, South Korea. The isolate, H9, was gram-negative and rod-shaped with a smooth surface and polar flagellum. Cells grew at 20-30℃, between pH 5.0 and 9.0, and in ASW-YP (Artificial Sea Water-Yeast extract, Peptone) media containing 1-5% (w/v) NaCl. The G+C content was 41.56 mol%. The predominant isoprenoid quinone in strain H9 was ubiquinone-8. The major fatty acids (>10%) were C_16:1ω7c (34.3%), C_16:0 (23.72%), and C_18:1ω7c (13.64%). Based on 16S rRNA gene sequencing, and biochemical and chemotaxonomic characterization, the strain was designated as Pseudoalteromonas sp. H9 (=KCTC23887). In liquid culture supplemented with 0.2% agar, the cell density and agarase activity reached a maximum level of OD = 4.32 (48 h) and OD = 3.87 (24 h), respectively. The optimum pH and temperature for the extracellular crude agarases of H9 were 7.0 and 40℃, respectively. Thin-layer chromatography analysis of the agarase hydrolysis products revealed that the crude agarases hydrolyze agarose into neoagarotetraose and neoagarohexaose. Therefore, the new agar-degrading strain, H9, can be applicable for the production of valuable neoagarooligosaccharides and for the complete degradation of agar in bio-industries.

• Journal of Life Science
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• v.25 no.11
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• pp.1273-1279
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• 2015

Agarase from a novel agar-degrading bacterium isolated from seawater in Namhae at Gyeongsangnamdo province of Korea was characterized. The SH-1 strain was selected from thousands of colonies on Marine agar 2216 media. Almost full 16S rRNA gene sequence of the agarolytic SH-1 strain showed 99% similarity with that of bacteria of Simiduia genus and named as Simiduia sp. SH-1. Agarase production was growth related, and activity was declined from stationary phase. Secreted agarase was prepared from culture media and characterized. It showed maximum activity of 698.6 units/L at pH 7.0 and 30℃ in 20 mM Tris-HCl buffer. Agarase activity decreased as the temperature increased from an optimum of 30℃, with 90% and 75% activity at 40℃ and 50℃, respectively. Agarase was not heat resistant. Slightly lower agarase activity was observed at pH 6.0 than at pH 7.0, without statistical difference, and 80% and 75% activity were observed at pH 5.0 and 8.0, respectively. Neoagarotetraose and neoagarobiose were the main final products of agarose, indicating that it is β-agarase. Simiduia sp. SH-1 and its β-agarase would be useful for the industrial production of neoagarotetraose and neoagarobiose, which have a whitening effect on skin, delaying starch degradation, and inhibiting bacterial growth.

• Journal of Life Science
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• v.32 no.4
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• pp.285-289
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• 2022

In this study, the growth characteristics of an agar-degrading bacterium isolated from seawater samples collected from Yeongheungdo, Incheon, and the characteristics of its agarase were analyzed. The 16S rRNA gene sequence of the isolated strain was 95% similar to that of the genus Agarivorans, and thus the isolated strain was named Agarivorans sp. HY-1. When Agarivorans sp. HY-1 was cultured in a marine broth 2216 medium at 27℃ and 250 rpm, it showed maximum growth on day 1 and showed maximum enzymatic activity on day 2. A crude enzyme solution was prepared from secreted agarase in the culture medium. The extracellular agarase of the Agarivorans sp. HY-1 strain showed maximal activity at 40℃ and pH 7.0 (20 mM Tris-HCl) with 591.91 U/l. The agarase exhibited relative activities of 64, 91, 100, 97, 89, and 60% at 20, 30, 40, 50, 60, and 70℃, respectively. At pH 5, 6, 7, and 8, the relative activities were 79, 95, 100, and 55%, respectively. Furthermore, the agarase exhibited >86% residual activity at 20, 30, and 40℃ for 2 hr and >44% residual activity at 50℃ after 2 hr. A TLC analysis confirmed that Agarivorans sp. HY-1 produced α-agarase. As the degradation products of α-agarase have anticancer and antioxidant effects, Agarivorans sp. HY-1 and its agarase may well prove useful.

• Journal of Microbiology and Biotechnology
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• v.30 no.11
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• pp.1659-1669
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• 2020

1,3-α-3,6-anhydro-L-galactosidase (α-neoagarooligosaccharide hydrolase) catalyzes the last step of agar degradation by hydrolyzing neoagarobiose into monomers, D-galactose, and 3,6-anhydro-L-galactose, which is important for the bioindustrial application of algal biomass. Ahg943, from the agarolytic marine bacterium Gayadomonas joobiniege G7, is composed of 423 amino acids (47.96 kDa), including a 22-amino acid signal peptide. It was found to have 67% identity with the α-neoagarooligosaccharide hydrolase ZgAhgA, from Zobellia galactanivorans, but low identity (< 40%) with the other α-neoagarooligosaccharide hydrolases reported. The recombinant Ahg943 (rAhg943, 47.89 kDa), purified from Escherichia coli, was estimated to be a monomer upon gel filtration chromatography, making it quite distinct from other α-neoagarooligosaccharide hydrolases. The rAhg943 hydrolyzed neoagarobiose, neoagarotetraose, and neoagarohexaose into D-galactose, neoagarotriose, and neoagaropentaose, respectively, with a common product, 3,6-anhydro-L-galactose, indicating that it is an exo-acting α-neoagarooligosaccharide hydrolase that releases 3,6-anhydro-L-galactose by hydrolyzing α-1,3 glycosidic bonds from the nonreducing ends of neoagarooligosaccharides. The optimum pH and temperature of Ahg943 activity were 6.0 and 20℃, respectively. In particular, rAhg943 could maintain enzyme activity at 10℃ (71% of the maximum). Complete inhibition of rAhg943 activity by 0.5 mM EDTA was restored and even, remarkably, enhanced by Ca²⁺ ions. rAhg943 activity was at maximum at 0.5 M NaCl and maintained above 73% of the maximum at 3M NaCl. K_m and V_max of rAhg943 toward neoagarobiose were 9.7 mg/ml and 250 μM/min (3 U/mg), respectively. Therefore, Ahg943 is a unique α-neoagarooligosaccharide hydrolase that has cold- and high-salt-adapted features, and possibly exists as a monomer.