Purification and characterization of alginate lyase from Sphingomonas sp. ZH0.

Alginate lyases degrade alginate in a beta-elimination reaction to produce oligosaccharides. Thus, alginate lyases are widely used in the food/pharmaceutical industries and are commercially valuable. In this study, four alginate lyase encoding genes were successfully cloned from Sphingomonas sp. ZH0. The expression systems of these alginate lyases were then constructed in Escherichia coli cells. The recombinant ZH0-I, ZH0-II, ZH0-III and ZH0-IV were purified from E. coli cells and were confirmed to be monomeric enzymes with molecular weights of approximately 91, 52, 67, and 113 kDa, respectively. The conditions for enzymes to have the highest specific lyase activities were 53.2 U/mg, 42 °C, pH 7.0 for ZH0-I, 103.9 U/mg, 47 °C, pH 6.5 for ZH0-II, 13.7 U/mg, 52 °C, pH 7.5 for ZH0-III, and 12.3 U/mg, 37 °C, pH 7.0 for ZH0-IV, respectively. These recombinant enzymes were stable over a pH range. Moreover, the enzymes were active in the absence of salt ions, and their activities were substantially reduced by the addition of HgCl. ZH0-I, ZH0-II and ZH0-III belong to endotype alginate lyases, while ZH0-IV is an exotype alginate lyase. All types could degrade both poly-β-d-mannuronate and poly-α-l-guluronate blocks, yielding alginate oligosaccharides as the main product. The K and V values were 0.51 mg/ml and 56.18 U/ml for ZH0-I, 0.47 mg/ml and 27.5 U/ml for ZH0-II, 0.55 mg/ml and 60.24 U/ml for ZH0-III, and 0.41 mg/ml and 5.53 U/ml for ZH0-IV, respectively. These features indicate that these alginate lyases are promising candidates for producing antioxidants from alginates in industrial applications.