Affinity purification of beta-amylases originating from plant using cyclomaltohexaose-immobilized Sepharose 6B in the presence of ammonium sulfate.

This paper reports a novel method of affinity purification of soybean and barley beta-amylase on cyclomaltohexaose-immobilized Sepharose. Until now, it has been shown that sweet potato beta-amylase can be purified using the above absorbent but beta-amylases from soybean and barley seeds cannot. We found that soybean and barley beta-amylase becomes adsorbed specifically on the above absorbent if it is in solution with 1 to 2 M ammonium sulfate, and the adsorbed enzyme can be easily eluted with a buffer containing no ammonium sulfate. Employing this procedure, soybean beta-amylase was demonstrated to be purified about 10-fold to homogeneity as judged from analysis of both a sodium dodecyl sulfate-gel electrophoresis and the specific activity, using a crude enzyme preparation (sp act 95 U/mg) as a starting material. The specific activity of this highly purified enzyme (950 U/mg) was almost the same as that of crystallized soybean beta-amylase at 37 degrees C.