An exotype alginate lyase in Sphingomonas sp. A1: overexpression in Escherichia coli, purification, and characterization of alginate lyase IV (A1-IV).
Sphingomonas sp. A1 (strain A1) cells contain three kinds of endotype alginate lyases [A1-I, A1-II, and A1-III], all of which are formed from a common precursor through posttranslational processing. In addition to these lyases, another type of lyase (A1-IV) that acts on oligoalginates exists in the bacterium. A1-IV was overexpressed in Escherichia coli cells through control of its gene under the T7 promoter. The expression level of the enzyme in E. coli cells was 8.6U/L-culture, which was about 270-fold higher than that in strain A1 cells. The enzyme was purified to homogeneity through three steps with an activity yield of 10.9%. The optimal pH and temperature, thermal stability, and mode of action of the purified enzyme were similar to those of the native enzyme from strain A1 cells. A1-IV exolytically degraded oligoalginates, which were produced from alginate through the reaction of A1-I, A1-II, or A1-III, into monosaccharides, indicating that the cooperative actions of these four enzymes cause the complete depolymerization of alginate in strain A1 cells.
Division of Food and Biological Science, Department of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
Osamu
O
Miyake
Wataru
W
Hashimoto
Kousaku
K
Murata
D000464:Alginates / Q000737:chemistry; D002240:Carbohydrate Sequence; D002855:Chromatography, Thin Layer; D004926:Escherichia coli / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000737:chemistry*; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D020578:Sphingomonas / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
D000464:Alginates; D007477:Ions; D011994:Recombinant Proteins; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase