Purification and characterization of a new agarase from a marine bacterium, Vibrio sp. strain JT0107.
A marine bacterial strain that decomposes the cell walls of some seaweeds, including a Laminaria sp. and Undaria pinnatifida, has been isolated from seawater. This strain has been classified to the genus Vibrio. One of the enzymes which the bacteria secreted into the culture medium was isolated and purified 45-fold from the culture fluid by a combination of ammonium sulfate precipitation and successive rounds of anion-exchange column chromatography. Purified protein migrated as a single band (M(r), 107,000) on sodium dodecyl sulfate-polyacrylamide gels. By amino acid sequence analysis, it was determined that this protein had a single N-terminal sequence that did not exhibit identity with the sequences of other agarases from marine bacteria. This novel enzyme was found to be an endo-type beta-agarase (EC 3.2.1.81) which hydrolyzes the beta-1,4 linkage of agarose to yield neoagarotetraose [O-3,6-anhydro-alpha-L-galactopyranosyl (1-->3)-O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro-alpha-L-galactopy ranosyl (1-->3)-D-galactose] and neoagarobiose [O-3,6-anhydro-alpha-L-galactopyranosyl (1-->3)-D-galactose] at a pH of around 8. The optimum temperature was 30 degrees C. This enzyme did not decompose sodium alginate or lambda-, iota-, or kappa-carrageenan. This enzyme may be of practical application in gene technology in the isolation of DNA fragments from agarose gels after electrophoresis.
Sugano
Y
Y
Life Science Research Laboratory, Japan Tobacco Inc., Kanagawa.
Terada
I
I
Arita
M
M
Noma
M
M
Matsumoto
T
T
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002240:Carbohydrate Sequence; D004247:DNA / Q000302:isolation & purification; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism; D005697:Galactosides; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides; D012623:Seawater; D012685:Sepharose / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014733:Vibrio / Q000201:enzymology* / Q000302:isolation & purification; D014871:Water Microbiology
D000596:Amino Acids; D004187:Disaccharides; D005697:Galactosides; D009844:Oligosaccharides; C081798:neoagarotetraose; C068544:neoagarobiose; D004247:DNA; D012685:Sepharose; D006026:Glycoside Hydrolases; C019624:agarase