Cloning, Expression, and Characterization of a Thermotolerant β-agarase from Simiduia sp. SH-4
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The gene coding for a thermotolerant β-agarase from an isolated Simiduia sp. SH-4 was cloned, recombinantly expressed, and characterized after purification. This gene was sequenced after cassette mediated polymerase chain reaction and composed of an open reading frame of 1,809 base pairs, encoding a protein of 66.2 kilodaltons comprising of 602 amino acid residues. The amino acids sequence showed 74% homology with β-agarase of Simiduia agarivorans. A new β-agarase gene corresponding to mature protein of 577 amino acids was recombinantly expressed and purified by chitin bead column to homogeneity. The maximal specific activity was 505.07 U/mg at 50oC in Tris/HCl (pH 6.0) buffer. Recombinant β-agarase hydrolyzed agar into neoagarotetraose (57%) and neoagarohexaose (43%). It generated products from melted and non-melted powder agar and agarose at 30-50oC, meaning cheap agar materials could be used with energy- and costsavings. Thus, recombinant β-agarase could be used for industrial production of neoagarotetraose and neoagarohexaose.
Keywordsβ-agarase cloning expression purification Simiduia sp.
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