Purification and characterization of a novel enzyme, alpha-neoagarooligosaccharide hydrolase (alpha-NAOS hydrolase), from a marine bacterium, Vibrio sp. strain JT0107.
A novel enzyme, alpha-neoagarooligosaccharide hydrolase (EC 3.2.1.-), which hydrolyzes the alpha-1,3 linkage of neoagarooligosaccharides to yield agaropentaose (O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro-alpha-L-galactopyranosyl (1-->3)-D-galactose], agarotriose [O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro- alpha-L-galactopyranosyl (1-->3)-D-galactose], agarobiose [O-beta-D-galactopyranosyl(1-->4)-3,6-anhydro-L-galactose], 3,6-anhydro-L-galactose, and D-galactose was isolated from the marine bacterium Vibrio sp. strain JT0107 and characterized. This enzyme was purified 383-fold from cultured cells by using a combination of ammonium sulfate precipitation, successive anion-exchange column chromatography, gel filtration, and hydroxyapatite chromatography, gel filtration, and hydroxyapatite chromatography. The purified protein gave a single band (M(r), 42,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estimation of the M(r) by the gel filtration method gave a value of 84,000, indicating that the enzyme is dimeric. Amino acid sequence analysis revealed it to have a single N-terminal sequence that has no sequence homology to any other known agarases. The optimum temperature and pH were 30 degrees C and 7.7, respectively. The Km and maximum rate of metabolism for neoagarobiose were 5.37 mM and 92 U/mg of protein, respectively.
Sugano
Y
Y
Seawater Science Research Laboratory, Japan Tobacco Inc., Kanagawa.
Kodama
H
H
Terada
I
I
Yamazaki
Y
Y
Noma
M
M
D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008386:Marine Biology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D012685:Sepharose / Q000378:metabolism; D017421:Sequence Analysis; D013379:Substrate Specificity; D014733:Vibrio / Q000201:enzymology*; D014871:Water Microbiology
D009844:Oligosaccharides; D012685:Sepharose; D006026:Glycoside Hydrolases; C090400:alpha-neoagarooligosaccharide hydrolase