Phenylboronic acid as a ligand for biospecific chromatography of serine proteinases.
Via attachment of p-(omega-aminoethyl)phenylboronic acid to CH-Sepharose in the presence of water-soluble carbodiimide, a new sorbent for the biospecific chromatography of serine proteinases was obtained. The sorbent was shown to be suitable for the purification of subtilisn, alpha-chymotrypsin and trupsin. It is assumed that the serine hydroxyl group at the active site of the enzyme forms, with the boronic acid moiety of the ligand, a structure that imitates transition enzyme--substrage complex. The presence of glycerol selectively improves the binding of serine proteinases, presumably because of stabilization of the tetrahedral state of the boron atom. Direct isolation of subtilisin from a Bacillus subtilis cultural filtrate on phenylboronic acid-containing sorbent gives a virtually homogenous enzyme (42-fold purification) in a nearly-quantitative yield.
D001412:Bacillus subtilis / Q000201:enzymology; D001897:Boronic Acids; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D002855:Chromatography, Thin Layer; D002918:Chymotrypsin / Q000302:isolation & purification; D010450:Endopeptidases / Q000302:isolation & purification*; D008024:Ligands; D013381:Subtilisins / Q000302:isolation & purification; D014357:Trypsin / Q000302:isolation & purification
D001897:Boronic Acids; D008024:Ligands; D010450:Endopeptidases; D013381:Subtilisins; D002918:Chymotrypsin; D014357:Trypsin