Overexpression in Escherichia coli, purification, and characterization of Sphingomonas sp A1 alginate lyases

A bacterium Sphingomonas sp. 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 algi nate lyases were constructed in Escherichia coli cells by controlling of th e lyase genes under T7 promoter and terminator. Expression levels of A1-I, A1-II, and A1-III in E. coli cells were 3.50, 3.04, and 2.13 kU/liter of cu lture, respectively, and were over 10-fold higher than those in Sphingomona s sp, Al cells. Purified A1-I, A1-II, and A1-III from E. coli cells were mo nomeric 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 beta-elimination reaction. A1-II preferred polyguluronate rather than po lymannuronate and released tri- and tetrasaccharides, which have unsaturate d uronyl residues at the nonreducing terminal, from alginate as the major f inal products. A1-I acted equally on both homopolymers and produced di- and trisaccharides as the final products. (C) 2000 Academic Press.