Overexpression in Escherichia coli,Purification, and Characterization of Sphingomonassp. A1 Alginate Lyases

A bacterium Sphingomonassp. A1 produces three kinds of alginate lyases [A1-I (66 kDa), A1-II (25 kDa), and A1-III (40 kDa)] from a single precursor, through posttranslational processing. Overexpression systems for these alginate lyases were constructed in Escherichia colicells by controlling of the lyase genes under T7 promoter and terminator. Expression levels of A1-I, A1-II, and A1-III in E. colicells were 3.50, 3.04, and 2.13 kU/liter of culture, respectively, and were over 10-fold higher than those in Sphingomonassp. A1 cells. Purified A1-I, A1-II, and A1-III from E. colicells were monomeric enzymes with molecular masses of 63, 25, and 40 kDa, respectively. The depolymerization pattern of alginate with A1-I and A1-II indicated that both enzymes cleaved the glycosidic bond of the polymer endolytically and by β-elimination reaction. A1-II preferred polyguluronate rather than polymannuronate and released tri- and tetrasaccharides, which have unsaturated uronyl residues at the nonreducing terminal, from alginate as the major final products. A1-I acted equally on both homopolymers and produced di- and trisaccharides as the final products.