|Budget Amount *help
¥1,800,000 (Direct Cost : ¥1,800,000)
Fiscal Year 1991 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1990 : ¥900,000 (Direct Cost : ¥900,000)
A novel type of sulfotransferase, which we discovered from Eubacterium A-44, a human intestinal bacterium, catalyzes the transfer of a sulfate group of phenyl sulfate ester to phenolic hydroxyl group to form a new sulfate ester. Using the enzyme, we investigated enzymatic sulfation of various phenol compounds and obtained the following results.
1. We succeeded enzymatic sulfation of tyrosine-containing peptide hormons such as CCK, VIP, IL-II, angiotensins, hirudin, gastrin, and enkephalin, and of silk fibroin. Hormonal activities of brain-gut peptides were remarkably increased, but those of other peptides were decreased by the sulfation of tyrosine residues. Sulfation of silk fibroin caused tlye increase in water-solubility. Furthermore, sulfated peptides became resistant against chymotryptic and peptidase digestion.
2. Catecholamines and tyramine were well sulfated by the enzyme reaction with comitant loss of the physiological activity. Polyphenols of plant origins such as flavones and
tannins were sulfated as well, with position specificity. Enzymatic sulfation made these polyphenoles soluble in water, which is supposed to be one of the detoxication mechanism in the body, by affecting intestinal absorption and fecal excretion. And phenolic antibiotics were also sulfated without significant loss of antibiotic activity.
3. The enzyme was purified to homogeneity and its enzymic properties were clarified : the enzyme is a metallo-protein containing magnesium ion, and the reaction proceeds according a ping pong bi bi mechanism. The relations between pKa values of substrate phenols and reaction velocity or optimal pH were elucidated.
4. Production of the enzyme was induced by phenyl sulfate esters, sulfate donor substrates, in the cultivation of Enbacterium. High yields of the enzyme from sonicated cells were obtained when grown in a glycine-containing medium. Covalently immobilized enzyme was prepared, which was more stable than the native enzyme, for the industrial use of the enzyme in order to synthesize O-sulfated tyrosine-containing peptides and position specific O-sulfated polyphenols.