• 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.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.31-32
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• 2005

Neoagaro-oligosaccharides are produced only by enzymatic degradation of agarose by ${\beta}-agarase.^{1)}$ Neoagaro-oligosaccharides inhibit the growth of bacteria, slow the rate of degradation of starch, are used as low-calorie additives to improve food quality, and have macrophage-stimulating activity. Furthermore, neoagarobiose is a rare reagent that has both moisturizing effect on skin and whitening effect on melanoma $cells.^{2)}$ An agar-degrading marine bacterium was isolated from the sea water at the northeast coast in Cheju island, Korea. The strain was gram negative, aerobic, and motile rod. The 16S rRNA of the strain had the closest match of 98% homology, with that from Agarivorans albus. On the basis of several phenotypic characters and a phylogenetic analysis, this strain was designated Agarivorans sp. JA-1. In solid agar plate, Agarivorans sp. JA-1 produced a diffusible agarase that caused agar softening around the colonies. Agarivorans sp. JA-1 was cultured for 36 hr in marine broth 2216 (Difco, USA) and the supernatant that containing an extracellular ${\beta}-agarase$ was prepared by centrifugation of culture media. The enzyme exhibited relatively strong activity at $40^{\circ}C$ and was stable up to $60^{\circ}C$. Using PCR primers derived from the ${\beta}-agarase$ gene of Vibrio sp., the gene encoding ${\beta}-agarase$ from Agarivorans sp. JA-1 was cloned and sequenced. The structural gene consists of 2931 bp encoding 976 amino acids with a predicted molecular weight of 107,360 Da. The deduced amino acid sequence showed 99% and 34% homology to $agaA^{2)}$ and $agaB^{2)}$ genes for ${\beta}-agarase$ from Vibrio sp., respectively. The expression plasmid for ${\beta}-agarase$ gene of Agarivorans sp. JA-1 is being constructed and the recombinant enzyme will be biochemically characterized.

• Journal of Microbiology and Biotechnology
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• v.28 no.5
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• pp.776-783
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• 2018

The agarase gene gaa16a was identified from a draft genome sequence of Gilvimarinus agarilyticus JEA5, an agar-utilizing marine bacterium. Recently, three agarase-producing bacteria, G. chinensis, G. polysaccharolyticus, and G. agarilyticus, in the genus Gilvimarinus were reported. However, there have been no reports of the molecular characteristics and biochemical properties of these agarases. In this study, we analyzed the molecular characteristics and biochemical properties of agarases in Gilvimarinus. Gaa16A comprised a 1,323-bp open reading frame encoding 441 amino acids. The predicted molecular mass and isoelectric point were 49 kDa and 4.9, respectively. The amino acid sequence of Gaa16A showed features typical of glycosyl hydrolase family 16 (GH16) ${\beta}$-agarases, including a GH16 domain, carbohydrate-binding region (RICIN domain), and signal peptide. Recombinant Gaa16A (excluding the signal peptide and carbohydrate-binding region, rGaa16A) was expressed as a fused protein with maltose-binding protein at its N-terminus in Escherichia coli. rGaa16A had maximum activity at $55^{\circ}C$ and pH 7.0 and 103 U/mg of specific activity in the presence of 2.5 mM $CaCl_2$. The enzyme hydrolyzed agarose to yield neoagarotetraose as the main product. This enzyme may be useful for industrial production of functional neoagaro-oligosaccharides.