Purification and characterization of an enzyme releasing lacto-N-biose from oligosaccharides with type 1 chain.
An enzyme specific for oligosaccharides with type 1 chain was purified 7,000-fold from the culture broth of Streptomyces sp. 142. The enzyme, lacto-N-biosidase, was induced and secreted into culture medium when the strain was cultured in the presence of porcine stomach mucin. The enzyme was purified by anion-exchange chromatography on Q Sepharose, cation-exchange chromatography on S Sepharose, fast protein liquid chromatography on a Mono S column, and gel filtration chromatography on TSK gel HW55S. To remove contaminating alpha-1,3/4-fucosidase and beta-N-acetylglucosaminidase, final purification was done by fast protein liquid chromatography on a Mono S column and affinity chromatography on N-acetylglucosamine agarose. The purified enzyme gave only one major protein band with an apparent M(r) of 60,000 on sodium dodesyl sulfate-polyacrylamide gel electrophoresis. The enzyme had maximum activity at pH 5.5 and was stable at the pH range of 4.0-10.0. Substrate specificity studies with oligosaccharides labeled with 2-aminopyridine showed that the enzyme specifically hydrolyzed lacto-N-tetraose and the N-acetyllactosamine type of triantennary sugar chain with the type 1 chain, but did not hydrolyze type 2 chain oligosaccharides or the type 1 chain oligosaccharides with fucose or sialic acid including lacto-N-fucopentaose I and II and alpha-2,3-sialyl lacto-N-tetraose. The enzyme released lacto-N-biose from asialofetuin, and almost all oligosaccharides in asialofetuin were found to have only type 2 chains. Sequential digestion of extended type 1 chain oligosaccharides with alpha-1,3/4-fucosidase and lacto-N-biosidase was possible.
Sano
M
M
Biotechnology Research Laboratories, Takara Shuzo Company, Limited, Shiga, Japan.
Hayakawa
K
K
Kato
I
I
D001212:Asialoglycoproteins / Q000378:metabolism; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D060748:Fetuins; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009077:Mucins; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature; D000509:alpha-Fetoproteins / Q000378:metabolism
D001212:Asialoglycoproteins; D003470:Culture Media; D060748:Fetuins; D009077:Mucins; D009844:Oligosaccharides; D000509:alpha-Fetoproteins; C017029:asialofetuin; D006026:Glycoside Hydrolases; C076748:lacto-N-biosidase