Application of Immobilization Method to Mechanistic Study and Continuous Procedure of Protein Refolding

(1)为了选择蛋白质再折叠介质，制备了两种不同类型的固定化枯草菌素BPN’。一种是由酶的氨基固定在琼脂糖珠上，另一种是由酶的羧基固定在琼脂糖珠上。通过对固定化枯草菌素进行反复变性/复性处理得到的定量再化制剂，发现两种固定化制剂均可用于培养基的选择。(2)通过对枯草菌素和热溶菌素定量复生性制剂的研究，发现2 M乙酸钾和丙酸钾等有机酸是最佳复生性培养基。这些溶剂对蛋清溶菌酶的再折叠效果不明显。(3)对于一个分子中含有3个二硫键的灰色链霉菌胰蛋白酶（Streptomyces griseus trypsin， SGT），琼脂糖珠无法获得定量的变性制备，可能是因为变性/变性处理涉及氧化还原反应。相反，将SGT固定在玻璃微珠上获得了令人满意的结果。(4)固定化苯并脒对SGT具有抑制作用，可显著提高SGT在溶液体系中的再折叠率。(5)这一事实使得利用固定化抑制剂设计连续和大规模的SGT再折叠成为可能。

(1) In order to select protein refolding media, two different types of immobilized subtilisin BPN' were prepared. One was immobilized on agarose beads by the amino groups of the enzyme and the other was immobilized on agarose beads by the carboxyl groups. Using the quantitatively renaturing preparations obtained by repeated denaturation/renaturation treatment of the immobilized subtilisin, it was revealed that both of two immobilized preparations were useful for media selection.(2) By use of the quantitatively renaturing preparations of subtilisin and thermolysin, organic acids such as 2 M potassium acetate and potassium propionate were found to be excellent media. These solvents were not at all effective to the refolding of hen egg-white Lysozyme.(3) In the case of Streptomyces griseus trypsin (SGT) that involves 3 disulfide bonds in a molecule, the quantitatively renaturing preparation could not be obtained with agarose beads probably because the denaturation/renaturation treatment involved the redox reaction. Instead, immobilization of SGT on glass beads brought about the satisfactory result.(4) In the presence of immobilized benzamidine that represents inhibitory effect to SGT, the refolding yield of SGT in solution system was considerably increased.(5) This fact made it possible to design a continuous and a large-scale refolding of SGT by use of the immobilized inhibitor.