Totsuka A, Fukazawa C
Genetic Engineering Laboratory, National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Ibaraki, Japan.
Protein Expr Purif. 1993 Aug;4(4):333-6. doi: 10.1006/prep.1993.1043.
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.
本文报道了一种在固定化环麦芽六糖的琼脂糖上亲和纯化大豆和大麦β-淀粉酶的新方法。到目前为止，已经表明可以使用上述吸附剂纯化甘薯β-淀粉酶，但不能纯化来自大豆和大麦种子的β-淀粉酶。我们发现，如果大豆和大麦β-淀粉酶处于含有1至2M硫酸铵的溶液中，它会特异性吸附在上述吸附剂上，并且吸附的酶可以很容易地用不含硫酸铵的缓冲液洗脱。采用此方法，以粗酶制剂（比活性95U/mg）为起始材料，通过十二烷基硫酸钠凝胶电泳分析和比活性判断，大豆β-淀粉酶被纯化至约10倍的纯度。这种高度纯化的酶（950U/mg）在37℃下的比活性与结晶大豆β-淀粉酶几乎相同。
