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Molecular cloning, characterization and enzymatic properties of a novel ?eta-agarase from a marine isolate Psudoalteromonas SP. AG52.

ABSTRACT: An agar-degrading Pseudoalteromonas sp. AG52 bacterial strain was identified from the red seaweed Gelidium amansii collected from Jeju Island, Korea. A ?-agarase gene which has 96.8% nucleotide identity to Aeromonas ?-agarase was cloned from this strain, and was designated as agaA. The coding region is 870 bp, encoding 290 amino acids and possesses characteristic features of the glycoside hydrolase family (GHF)-16. The predicted molecular mass of the mature protein was 32 kDa. The recombinant ?-agarase (rAgaA) was overexpressed in Escherichia coli and purified as a fusion protein. The optimal temperature and pH for activity were 55 °C and 5.5, respectively. The enzyme had a specific activity of 105.1 and 79.5 unit/mg toward agar and agarose, respectively. The pattern of agar hydrolysis demonstrated that the enzyme is an endo-type ?-agarase, producing neoagarohexaose and neoagarotetraose as the final main products. Since, Pseudoalteromonas sp. AG52 encodes an agaA gene, which has greater identity to Aeromonas ?-agarase, the enzyme could be considered as novel, with its unique bio chemical characteristics. Altogether, the purified rAgaA has potential for use in industrial applications such as development of cosmetics and pharmaceuticals.

SUBMITTER: Oh C

PROVIDER: S-EPMC3769764 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Molecular cloning, characterization and enzymatic properties of a novel βeta-agarase from a marine isolate Psudoalteromonas SP. AG52.

Oh Chulhong C Nikapitiya Chamilani C Lee Youngdeuk Y Whang Ilson I Kang Do-Hyung DH Heo Soo-Jin SJ Choi Young-Ung YU Lee Jehee J

Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology] 20101001 4

An agar-degrading Pseudoalteromonas sp. AG52 bacterial strain was identified from the red seaweed Gelidium amansii collected from Jeju Island, Korea. A β-agarase gene which has 96.8% nucleotide identity to Aeromonas β-agarase was cloned from this strain, and was designated as agaA. The coding region is 870 bp, encoding 290 amino acids and possesses characteristic features of the glycoside hydrolase family (GHF)-16. The predicted molecular mass of the mature protein was 32 kDa. The recombinant β- ...[more]

PMID: 24031567

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Project description:Lysoplasmalogen (LyPls)-specific phospholipase D (LyPls-PLD) is an enzyme that catalyses the hydrolytic cleavage of the phosphoester bond of LyPls, releasing ethanolamine or choline, and 1-(1-alkenyl)-sn-glycero-3-phosphate (lysoplasmenic acid). Little is known about LyPls-PLD and metabolic pathways of plasmalogen (Pls). Reportedly, Pls levels in human serum/plasma correlate with several diseases such as Alzheimer's disease and arteriosclerosis as well as a variety of biological processes including apoptosis and cell signaling. We identified a LyPls-PLD from Thermocrispum sp. strain RD004668, and the enzyme was purified, characterized, cloned, and expressed using pET24a(+)/Escherichia coli with a His tag. The enzyme's preferred substrate was choline LyPls (LyPlsCho), with only modest activity toward ethanolamine LyPls. Under optimum conditions (pH 8.0 and 50 °C), steady-state kinetic analysis for LyPlsCho yielded Km and kcat values of 13.2 μm and 70.6 s-1, respectively. The ORF of LyPls-PLD gene consisted of 1005 bp coding a 334-amino-acid (aa) protein. The deduced aa sequence of LyPls-PLD showed high similarity to those of glycerophosphodiester phosphodiesterases (GDPDs); however, the substrate specificity differed completely from those of GDPDs and general phospholipase Ds (PLDs). Structural homology modeling showed that two putative catalytic residues (His46, His88) of LyPls-PLD were highly conserved to GDPDs. Mutational and kinetic analyses suggested that Ala55, Asn56, and Phe211 in the active site of LyPls-PLD may participate in the substrate recognition. These findings will help to elucidate differences among LyPls-PLD, PLD, and GDPD with regard to function, substrate recognition mechanism, and biochemical roles.Data accessibilityThermocrispum sp. strain RD004668 and its 16S rDNA sequence were deposited in the NITE Patent Microorganisms Depositary (NPMD; Chiba, Japan) as NITE BP-01628 and in the DDBJ database under the accession number AB873024. The nucleotide sequences of the 16S rDNA of strain RD004668 and the LyPls-PLD gene were deposited in the DDBJ database under the accession numbers AB873024 and AB874601, respectively.EnzymeEC number EC 3.1.4.4.

Dataset Information

Molecular cloning, characterization and enzymatic properties of a novel ?eta-agarase from a marine isolate Psudoalteromonas SP. AG52.

Publications

Molecular cloning, characterization and enzymatic properties of a novel βeta-agarase from a marine isolate Psudoalteromonas SP. AG52.

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