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.
Miyake
Osamu
O
Division of Food and Biological Science, Department of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
Hashimoto
Wataru
W
Murata
Kousaku
K
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