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 alginate lyases were constructed in Escherichia coli cells by controlling of the 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 culture, respectively, and were over 10-fold higher than those in Sphingomonas sp. A1 cells. Purified A1-I, A1-II, and A1-III from E. coli cells 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 beta-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.
Yoon
H J
HJ
Research Institute for Food Science, Kyoto University, Uji, 611-0011, Japan.
Hashimoto
W
W
Miyake
O
O
Okamoto
M
M
Mikami
B
B
Murata
K
K
D000464:Alginates / Q000737:chemistry*; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D020539:Sequence Analysis, Protein; D020578:Sphingomonas / Q000737:chemistry*; D013379:Substrate Specificity; D013696:Temperature
D000464:Alginates; D006603:Hexuronic Acids; D020723:Glucuronic Acid; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase