The development and application of a new affinity partitioning system for enzyme isolation and purification.
A reactive water-soluble polymer was synthesized by copolymerizing N-isopropylacrylamide and glycidyl acrylate. The reactive polymer could react with the amino groups of enzymes/proteins or other ligands to form an affinity polymer. As a model, the reactive polymer was allowed to react with paraaminobenzamidine, a strong trypsin inhibitor. The affinity polymer could easily form an aqueous two-phase system with either dextran or pullulan, and the phase diagram was compared favorably to that of the well-known polyethylene glycol-dextran system. Once trypsin was attracted to the affinity polymer dominant phase, the enzyme could be dissociated from the polymer at low pH. Owing to the N-isopropylacrylamide units, the affinity polymer could be isolated from the solution by precipitation at a low level of ammonium sulfate. The enzyme recovery was always greater than 50%, and the affinity polymer could be reused in several cycles of affinity partitioning and recovery.
Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec.
A L
AL
Nguyen
J H
JH
Luong
D000178:Acrylamides; D000179:Acrylates; D000588:Amines; D001550:Benzamidines; D002846:Chromatography, Affinity / Q000379:methods*; D003911:Dextrans; D004798:Enzymes / Q000302:isolation & purification*; D004852:Epoxy Compounds; D005936:Glucans; D011108:Polymers / Q000138:chemical synthesis; D012995:Solubility; D014357:Trypsin / Q000302:isolation & purification; D014361:Trypsin Inhibitors; D014867:Water
D000178:Acrylamides; D000179:Acrylates; D000588:Amines; D001550:Benzamidines; D003911:Dextrans; D004798:Enzymes; D004852:Epoxy Compounds; D005936:Glucans; D011108:Polymers; D014361:Trypsin Inhibitors; D014867:Water; C062422:2,3-epoxypropyl acrylate; C006500:4-aminobenzamidine; C009109:pullulan; C067295:N-isopropylacrylamide; D014357:Trypsin