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Int. J. Mol. Sci. 2015, 16(3), 5590-5603; doi:10.3390/ijms16035590

Extracellular Production of a Novel Endo-β-Agarase AgaA from Pseudomonas vesicularis MA103 that Cleaves Agarose into Neoagarotetraose and Neoagarohexaose

1
Department of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung 202, Taiwan
2
Department of Food Science, National Taiwan Ocean University, Keelung 202, Taiwan
3
Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung 202, Taiwan
*
Authors to whom correspondence should be addressed.
Academic Editor: Bing Yan
Received: 3 February 2015 / Revised: 3 March 2015 / Accepted: 4 March 2015 / Published: 11 March 2015
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
View Full-Text   |   Download PDF [3207 KB, uploaded 11 March 2015]   |  

Abstract

The gene agaA, of the isolated marine bacterium Pseudomonas vesicularis MA103, comprised 2958-bp nucleotides encoding a putative agarase AgaA of 985 amino acids, which was predicted to contain a signal peptide of 29 amino acids in the N-terminus, a catalytic domain of glycoside hydrolase 16 (GH16) family, a bacterial immunoglobulin group 2 (Big 2), and three carbohydrate binding modules 6 (CBM 6). The gene agaA was cloned and overexpressed in Escherichia coli, and the optimum temperatures for AgaA overexpression were 16, 20 and 24 °C. The agaA was cloned without its signal peptide for cytosolic production overexpression, whereas it was cloned with the heterologous signal peptide PelB and its endogenous signal peptide for periplasmic and extracellular productions, respectively. Extracellular and periplasmic rAgaA showed greater activity than that of cytosolic rAgaA, indicating that membrane translocation of AgaA may encourage proper protein folding. Time-course hydrolysis of agarose by rAgaA was accomplished and the products were analyzed using thin layer chromatography and matrix-assisted laser desorption inoization-time of flight mass spectrometry, indicating that AgaA from P. vesicularis was an endo-type β-1,4 agarase that cleaved agarose into neoagarotetraose and neoagarohexaose as the final products. View Full-Text
Keywords: agar; agarase; neoagaro-oligosaccharides; Pseudomonas vesicularis; osmotic shock; extracellular agar; agarase; neoagaro-oligosaccharides; Pseudomonas vesicularis; osmotic shock; extracellular
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Hsu, P.-H.; Wei, C.-H.; Lu, W.-J.; Shen, F.; Pan, C.-L.; Lin, H.-T.V. Extracellular Production of a Novel Endo-β-Agarase AgaA from Pseudomonas vesicularis MA103 that Cleaves Agarose into Neoagarotetraose and Neoagarohexaose. Int. J. Mol. Sci. 2015, 16, 5590-5603.

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