• Journal of Life Science
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• v.31 no.3
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• pp.356-365
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• 2021

Recently, we sequenced the entire genome of a freshwater agar-degrading bacterium Cellvibrio sp. KY-GH-1 (KCTC13629BP) to explore genetic information encoding agarases that hydrolyze agarose into monomers 3,6-anhydro-L-galactose (L-AHG) and D-galactose. The KY-GH-1 strain appeared to possess nine β-agarase genes and two α-neoagarobiose hydrolase (α-NABH) genes in a 77-kb agarase gene cluster. Based on these genetic information, the KY-GH-1 strain-caused agarose degradation into L-AHG and D-galactose was predicted to be initiated by both endolytic GH16 and GH86 β-agarases to generate NAOS (NA4/NA6/NA8), and further processed by exolytic GH50 β-agarases to generate NA2, and then terminated by GH117 α-NABHs which degrade NA2 into L-AHG and D-galactose. More recently, by employing E. coli expression system with pET-30a vector we obtained three recombinant His-tagged GH50 family β-agarases (GH50A, GH50B, and GH50C) derived from Cellvibrio sp. KY-GH-1 to compare their enzymatic properties. GH50A β-agarase turned out to have the highest exolytic β-agarase activity among the three GH50 isozymes, catalyzing efficient NA2 production from the substrate (agarose, NAOS or AOS). Additionally, we determined that GH117A α-NABH, but not GH117B α-NABH, could potently degrade NA2 into L-AHG and D-galactose. Sequentially, we examined the enzymatic characteristics of GH50A β-agarase and GH117A α-NABH, and assessed their efficiency for NA2 production from agarose and for production of L-AHG and D-galactose from NA2, respectively. In this review, we describe the benefits of recombinant GH50A β-agarase and GH117A α-NABH originated from Cellvibrio sp. KY-GH-1, which may be useful for the enzymatic hydrolysis of agarose for mass production of L-AHG and D-galactose.

• Journal of Life Science
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• v.15 no.6 s.73
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• pp.884-888
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• 2005

An agar-degrading bacterium was isolated from north-eastern sea of Jeju island and cultured in marine agar 2216 media. Biochemical and morphologicl characteristics and 165 rRNA gene revealed that isolated strain was member of Agarivorans genus, and named Agarivorans sp. JA-1. Agarase was produced as growth-related and expressed regardless of agar presence. Optimal pH was 8 at 50 mM Clycine-NaOH buffer, and activity was maximum at $40^{\circ}C$E Enzymatic activity was maintained over $80\%$ at $60^{\circ}C$t and $70\%$ at $80^{\circ}C$ which is thermotolerant. Hence isolated novel Agarivorans sp. JA-1 strain and its beta-agarase could be used for the production of functional oligosaccharide from agar in solution state.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.315-315
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• 2005

• Proceedings of the Korean Society of Life Science Conference
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• 2005.09a
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• pp.61-61
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• 2005

• Proceedings of the Korean Society of Life Science Conference
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• 2005.04a
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• pp.156-156
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• 2005

• Proceedings of the Korean Society of Life Science Conference
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• 2006.09a
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• pp.39.1-39.1
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• 2006

• Journal of Microbiology and Biotechnology
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• v.19 no.10
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• pp.1197-1200
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• 2009

The whitening effect, tyrosinase inhibition, and cytotoxicity of neoagarotetraose were measured after its purification from hydrolyzed agar by gel filtration chromatography. In melanoma B16F10 cells, the melanin content of neoagarotetraose-treated cells was the same as that treated by kojic acid or arbutin. In addition, tyrosinase of melanoma cells was strongly inhibited by neoagarotetraose at a concentration of $1{\mu}g/ml$ and similarly inhibited at 10 and $100{\mu}g/ml$ compared with those by arbutin or kojic acid. The activity of mushroom tyrosinase showed a 38% inhibition by neoagarotetraose at $1{\mu}g/ml$, and this inhibitory effect was more efficient than that by kojic acid. Neoagarotetraose revealed a similar $IC_{50}$ (50% inhibition concentration) value for mushroom tyrosinase as that by kojic acid. These data suggest that the neoagarotetraose generated from agar by recombinant $\beta$-agarase might be a good candidate as a cosmetic additive for the whitening effect.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.48 no.1
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• pp.58-63
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• 2015

The chemical and rheological properties of natural and enzymatically hydrolyzed porphyran isolated from Pyropia yezoensis were investigated. The enzymatic hydrolysate was prepared by hydrolysis of porphyran using ${\beta}$-agarase followed by fractionation based on molecular weight (>300 kDa (Fr-1), 100-300 kDa (Fr-2), 10-100 kDa (Fr-3) and 1-10 kDa (Fr-4) using an ultrafiltration membrane. Each hydrolysate fraction consisted mainly of galactose (42.7-57.5%), 3,6-anhydro galactose (6.5-15.1%) and ester sulfate (8.6-14.1%). The sulfate content of the enzymatically hydrolyzed fractions decreased with an increase in molecular weight, whereas the 3,6-anhydro galactose content increased significantly. The rheological behavior of porphyran and enzymatically hydrolyzed porphyran solutions demonstrated a pseudoplastic behavior, which agrees with the Herschel-Bulkley model. The effect of temperature on the viscosity of the porphyrans and hydolysate fractions were measured and modeled using the Arrhenius equation. The activation energy of the porphyrans and enzymatically hydrolyzed porphyran (Fr-1) increased from 12.30 to 20.29 kJ/mol and 9.06 to 23.84 kJ/mol, respectively with increasing concentrations from 3% to 7%. These data indicate that the extent of the apparent viscosity of porphyran and enzymatically hydrolyzed porphyran are influenced by both temperature and concentration.

• Journal of Microbiology and Biotechnology
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• v.29 no.4
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• pp.625-632
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• 2019

The unified saccharification and fermentation (USF) system was developed for direct production of ethanol from agarose. This system contains an enzymatic saccharification process that uses three types of agarases and a fermentation process by recombinant yeast. The $pGMF{\alpha}-HGN$ plasmid harboring AGAH71 and AGAG1 genes encoding ${\beta}-agarase$ and the NABH558 gene encoding neoagarobiose hydrolase was constructed and transformed into the Saccharomyces cerevisiae 2805 strain. Three secretory agarases were produced by introducing an S. cerevisiae signal sequence, and they efficiently degraded agarose to galactose, 3,6-anhydro-L-galactose (AHG), neoagarobiose, and neoagarohexose. To directly produce ethanol from agarose, the S. cerevisiae $2805/pGMF{\alpha}-HGN$ strain was cultivated into YP-containing agarose medium at $40^{\circ}C$ for 48 h (for saccharification) and then $30^{\circ}C$ for 72 h (for fermentation). During the united cultivation process for 120 h, a maximum of 1.97 g/l ethanol from 10 g/l agarose was produced. This is the first report on a single process containing enzymatic saccharification and fermentation for direct production of ethanol without chemical liquefaction (pretreatment) of agarose.

• Microbiology and Biotechnology Letters
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• v.42 no.4
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• pp.324-330
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• 2014

A marine bacterial strain producing agar-degrading enzymes was isolated from a mud flat in Jeboo-do (Korea) using a selective artificial sea water (ASW) agar plate containing agar as the sole carbon source. The isolate, designated as SH-1, was gram-negative, aerobic, and motile with single polar flagellum. 16S rRNA gene sequence similarity analysis showed the isolate SH-1 had the highest homology (96.5%) to marine bacterium Neiella marina J221. Cells could grow at $28-37^{\circ}C$ but not at $42^{\circ}C$, and the agarase activity of the cell culture supernatant was higher when grown at $28^{\circ}C$ than when grown at $37^{\circ}C$. Cells could grow when concentrations of 1-5% (w/v) NaCl were added to the growth media with the best growth observed at 3% NaCl, and the agardegrading enzyme activity of the cell culture supernatant was best when grown at 3% NaCl-containing growth media under the conditions we examined. The crude enzyme prepared from 48-h culture broth of strain SH-1 exhibited an optimum pH and temperature for agar-degrading activity at 7.0 and $40^{\circ}C$, respectively. Zymogram analysis of the crude supernatant and cell extract showed that strain SH-1 produced at least 3 agar-degrading enzymes with molecular weights of 15, 35, and 52 KD. Thinlayer chromatography (TLC) analysis also suggested that HS-1 produces ${\beta}$-agarase to degrade agarose to neoagarooligosaccharides.