Construction of a Genetic Engineering Strain、Recombinant Expression and in Vivo Refolding of a Novel β-Agarase

Abstract: Agarases degrade agar or agarose and are classified into two groups according to the modes of action:α?andβ-agarases, which hydrolyzeα-1, 3 linkages andβ-1, 4 linkages in agarose, respectively. Most known agarases areβ-agarase, with the exception of threeα-agarases, and more frequently,β-agarases hydrolyze agar or agarose yielding neoagarobiose, -tetraose or -hexaose as the main products. Agarases have many applications in food, cosmetic, and medical industries for the production of oligosaccharides from agar. A novelβ-agarase AgaB studied in this paper has been suggested to represent a new family of glycoside hydrolase. AgaB was cloned from Pseudoalteromonas sp. CY24. Structurally, agaB gene is not significantly similar to any known genes, including glycoside hydrolase ones being identified currently. Functionally, AgaB hydrolyzes agarose or agar, producing neoagarooctaose and neoagarodecaose as the major end products, whereas most ofβ-agarases hydrolyze agarose or agar, mainly producing low degree of neoagaro-oligosaccharides. Thus, AgaB has large potential value in either basic research or application in food, medical and some other fields.Although AgaB has been expressed in E.coli, the protocol of expression and purification was time-consuming and costly. Only 2~3mg protein was obtained from 1L fermentation supernatant. Consequently, agaB gene was cloned into …
Key words: β-agarases; temperature controlled vector; inclusion bodies; refolding ; immobilized metal affinity chromatography (IMAC)

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