• Journal of Applied Biological Chemistry
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• v.53 no.2
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• pp.105-107
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• 2010

To overcome the limitation of E. coli expression system such as inclusion body formation and disulfide bond failure, we tried to express the heterologous protein as a secreted form. We adopted agarase signal peptide (ASP; 23 amino acid residues) from Agarivorans albus YKW-34 which is one of marine bacteia. When we used ASP to express $\beta$-agarase, about 42% activity was detected in media.

• Journal of Microbiology and Biotechnology
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• v.21 no.8
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• pp.818-821
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• 2011

An agar-degrading bacterium was isolated from the guts of spiny turban shells. It was identified as a Pseudoalteromonas species and named Pseudoalteromonas sp. JYBCL 1. The viscosity of the inoculated agar medium decreased by more than 60% after 20 h cultivation. The agarase produced by the isolate had optimal activities at $35^{\circ}C$ and pH 7. The enzyme had extremely strong resistance to ionic stress compared with other known agarases. Its molecular mass was estimated at about 60 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The agarase could saccharify Gelidium amansii directly, with an efficiency about half that compared with agar saccharification.

• KSBB Journal
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• v.14 no.5
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• pp.572-577
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• 1999

A marine bacterium with highly effective agar degrading activity was ioslated from the southern sea of Korea (Chonnam, YoChon) and identified as Cytophaga sp. and named as Cytophaga sp. AYK301. This strain produced an extracellular agarase which had a high activity with agar. The optimum culture conditions for the production of agarase have been determined. For the increase of agarase productivity, 0.2% agar, 0.3% beef extract, and 0.05% NH$_4$NO$_3$ were used as carbon, organic and inorganic nitrogen source, respectively. The optimal initial pH, NaCl, culture time and temperature for the agar degrading activity were 7.5, 7.0%, 36 hr and $25^{\circ}C$, respectively. In the optimal conditions, the agarase production was increased up to more than 4.0 folds as compared to that by the basal medium.

• Journal of Microbiology and Biotechnology
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• v.19 no.3
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• pp.257-264
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• 2009

A $\beta$-agarase gene, agaB34, was functionally cloned from the genomic DNA of a marine bacterium, Agarivorans albus YKW-34. The open reading frame of agaB34 consisted of 1,362 bp encoding 453 amino acids. The deduced amino acid sequence, consisting of a typical N-terminal signal peptide followed by a catalytic domain of glycoside hydrolase family 16 (GH-16) and a carbohydrate-binding module (CBM), showed 37-86% identity to those of agarases belonging to family GH-16. The recombinant enzyme (rAgaB34) with a molecular mass of 49 kDa was produced extracellularly using Escherichia coli $DH5{\alpha}$ as a host. The purified rAgaB34 was a $\beta$-agarase yielding neoagarotetraose (NA4) as the main product. It acted on neoagarohexaose to produce NA4 and neoagarobiose, but it could not further degrade NA4. The maximal activity of rAgaB34 was observed at $30^{\circ}C$ and pH 7.0. It was stable over pH 5.0-9.0 and at temperatures up to $50^{\circ}C$. Its specific activity and $k_{cat}/K_m$ value for agarose were 242 U/mg and $1.7{\times}10^6/sM$, respectively. The activity of rAgaB34 was not affected by metal ions commonly existing in seawater. It was resistant to chelating reagents (EDTA, EGTA), reducing reagents (DTT, $\beta$-mercaptoethanol), and denaturing reagents (SDS and urea). The E. coli cell harboring the pUC18-derived agarase expression vector was able to efficiently excrete agarase into the culture medium. Hence, this expression system might be used to express secretory proteins.

• Journal of Life Science
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• v.17 no.1 s.81
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• pp.70-75
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• 2007

A novel agar-degrading bacterium SL-5 was isolated from seashore of Homigot at Kyung-Buk province, and cultured in marine broth 2216 media. The bacterium SL-5 was identified as Thalassomonas genus by 16S rDNA sequencing with 96% identity. Growth rate was faster at $27^{\circ}C$ than at $37^{\circ}C$ and agarase was produced as growth-related. The optimum pH of the enzyme activity was 7.0 and the optimum temperature for the reaction was $40^{\circ}C$. Although the enzyme had no thermostability, the enzyme activity was remained over 80% at $60^{\circ}C$. The enzyme hydrolyzed neoagarohexaose to yield neoagarobiose as the main product, indicating that the enzyme is $\beta-agarase$. Thus, the enzyme would be useful for the industrial production of neoagarobiose.

• Journal of Microbiology and Biotechnology
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• v.3 no.1
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• pp.31-38
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• 1993

A bacterial strain KY-l isolated from sewage was able to produce an extracellular agarase(agarose 4-glycanohydrolase. EC 3.2.1.81). The strain KY-1 was identified as Cytophaga fermentans subsp. agarovorans based on its morphological and physiological characteristics. The agarase was purified by ammonium sulfate precipitation followed by DEAE-Sephadex A-50. Bio-Gel P-100. and CM-Cellulose column chromatography. The molecular weight of the purified enzyme was 24 kDa by SDS-polyacrylamide gel electrophoresis. The optimum temperature and pH for the enzyme activity were 30^{circ}C and 7.5, respectively. The enzyme activity was significantly inhibited in the presence of 0.1 mM $HgCl_2$. whereas it was elevated 3 times by $MnSO_4$ at 1 mM concentration. The Km value and Vmax were 16.67 mg/ml and 3.77 unit/ml.min. The agarase gene was cloned into Escherichia coli MC1061 using the plasmid vector pBR322. A 1.4 Kb DNA fragment of PstI-digested chromosomal DNA of C. fermentans KY-l was inserted into the PstI site of pBR322. expressed in the E. coli. and up to 60% of the total enzyme was extracellularly secreted. Enzymatic properties of the extracellular agarases produced by both the transformant and the donor were very similar in terms of optimal pH and temperature.

• Journal of Microbiology and Biotechnology
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• v.21 no.11
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• pp.1116-1122
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• 2011

In this study, site-directed mutagenesis was performed on the ${\beta}$-agarase AgaA gene from Zobellia galactanivorans to improve its catalytic activity and thermostability. The activities of three mutant enzymes, S63K, C253I, and S63K-C253I, were 126% (1,757.78 U/mg), 2.4% (33.47 U/mg), and 0.57% (8.01 U/mg), respectively, relative to the wild-type ${\beta}$-agarase AgaA (1,392.61 U/mg) at $40^{\circ}C$. The stability of the mutant S63K enzyme was 125% of the wild-type up to $45^{\circ}C$, where agar is in a sol state. The mutant S63K enzyme produced 166%, 257%, and 220% more neoagarohexaose, and 230%, 427%, and 350% more neoagarotetraose than the wild-type in sol, gel, and nonmelted powder agar, respectively, at $45^{\circ}C$ over 24 h. The mutant S63K enzyme produced 50% more neoagarooligosaccharides from agar than the wild-type ${\beta}$-agarase AgaA from agarose under the same conditions. Thus, mutant S63K ${\beta}$-agarase AgaA may be useful for the production of functional neoagarooligosaccharides.

• Journal of Microbiology and Biotechnology
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• v.22 no.12
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• pp.1621-1628
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• 2012

The agar-degrading bacterium GNUM-1 was isolated from the brown algal species Sargassum serratifolium, which was obtained from the West Sea of Korea, by using the selective artificial seawater agar plate. The cells were Gram-negative, $0.5-0.6{\mu}m$ wide and $2.0-2.5{\mu}m$ long curved rods with a single polar flagellum, forming nonpigmented, circular, smooth colonies. Cells grew at $20^{\circ}C-37^{\circ}C$, between pH 5.0 and 9.0, and at 1-10% (w/v) NaCl. The DNA G+C content of the GNUM-1 strain was 45.5 mol%. The 16S rRNA sequence of the GNUM-1 was very similar to those of Alteromonas stellipolaris LMG 21861 (99.86% sequence homology) and Alteromonas addita $R10SW13^T$(99.64% sequence homology), which led us to assign it to the genus Alteromonas. It showed positive activities for agarase, amylase, gelatinase, alkaline phosphatase, esterase (C8), lipase (C14), leucine arylamidase, valine arylamidase, ${\alpha}$-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, ${\alpha}$-galactosidase, ${\beta}$-galactosidase, ${\beta}$-glucosidase, catalase, and urease. It can utilize citrate, malic acid, and trisodium citrate. The major fatty acids were summed feature 3 (21.5%, comprising $C_{16:1}{\omega}7c/iso-C_{15:0}$ 2-OH) and C16:0 (15.04%). On the basis of the variations in many biochemical characteristics, GNUM-1 was considered as unique and thus was named Alteromonas sp. GNUM-1. It produced the highest agarase activity in modified ASW medium containing 0.4% sucrose, but lower activity in rich media despite superior growth, implying that agarase production is tightly regulated and repressed in a rich nutrient condition. The 30 kDa protein with agarase activity was identified by zymography, and this report serves as the very first account of such a protein in the genus Alteromonas.

• Journal of Life Science
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• v.29 no.11
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• pp.1173-1178
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• 2019

The purpose of this study was to isolate an agar-degrading marine bacterium and characterize its agarase. Bacterium BK-1, from Gwanganri Beach at Busan, Korea, was isolated on Marine 2216 agar medium and identified as Agarivorans sp. BK-1 by 16S rRNA gene sequencing. The extracellular agarase, characterized after dialysis of culture broth, showed maximum activity at pH 6.0 and $50^{\circ}C$ in 20 mM Tris-HCl buffer. Relative activities at 20, 30, 40, 50, 60, and $70^{\circ}C$ were 67, 93, 97, 100, 58, and 52%, respectively. Relative activities at pH 5, 6, 7, and 8 were 59, 100, 95, and 91%, respectively. More than 90% of the activity remained after a 2 hr exposure to 20, 30, or $40^{\circ}C$; about 60% of the activity remained after a 2 hr exposure to $50^{\circ}C$. Almost all activity was lost after exposure to 60 or $70^{\circ}C$ for 30 min. Zymography revealed three agarases with molecular weights of 110, 90, and 55 kDa. Agarose was degraded to neoagarobiose (46.8%), neoagarotetraose (39.7%), and neoagarohexaose (13.5%), confirming the agarase of Agarivorans sp. BK-1 as a ${\beta}$-agarase. The neoagarooligosaccharides generated by this agarase could be used for moisturizing, bacterial growth inhibition, skin whitening, food treatments, cosmetics, and delaying starch degradation.

• KSBB Journal
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• v.14 no.1
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• pp.96-102
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• 1999

Marine bacterium Bacillus cereus ASK202 produced an extracellular agarase (E.C.3.2.1.81) which showed a high level of enzyme activity in the presence of agar and agarose. In the optimal culture conditions, the agarase production increased 7.7 folds compared with the one obtained from the basal medium. Agarase production reached upto 160 units/L after 24hr of cultivation in a modified marine medium at $25^{\circ}C$. The degree of purification increased 31.5 folds with 27.8% yield through freeze drying, DEAE Sepharose CL-6B and Superose 6HR 10/30 column chromatography. The molecular weight of the purified agarase was determined to be 90,000 daltons by gel-permeation filteration. Optimal temperature and pH for the enzyme activity were $40^{\circ}C$ and 7.8, respectively. The enzyme was stable up to $50^{\circ}C$ and at a broad pH range of 5.0-10.0. The $\beta$-agarase was activated by $Zn(NO_3)_2$, and was inhibited by $CuSO_4$ and $SnCl_2$. The Km and Vmax values of this enzyme for agarose as a substrate was $2.4mg/m\ell$ and 13.6 mg/m$\ell$, respectively.