Porto Tatiana Souza, Pessôa-Filho Pedro Alcântara, Neto Benício Barros, Filho José Luiz Lima, Converti Attilio, Porto Ana Lúcia Figueiredo, Pessoa Adalberto
Department of Biochemical and Pharmaceutical Technology, University of São Paulo, 05508-000 Sio Paulo, SP, Brazil.
J Ind Microbiol Biotechnol. 2007 Aug;34(8):547-52. doi: 10.1007/s10295-007-0230-8.
In order to reduce the toxicity of Clostridium perfringens fermentation broths used in vaccine preparation, we developed two-phase aqueous systems for removal of toxin-activating proteases. Removal of the proteases inhibits the conversion of protoxin to active toxin. In order to establish the conditions under which the phase separation occurs, binodal curves, formed by poly(ethylene glycol) (PEG) and sodium citrate, were investigated at different values of pH and PEG molar mass. A 2(4)-experimental design was used to evaluate the influence of PEG molar mass and concentration, citrate concentration and pH on protease partition coefficient, removal.factor and protease removal yield. It has been found that simultaneous increase in PEG molar mass and decrease in citrate concentration remarkably improved the removal factor, whereas the protease removal yield showed an opposite trend. The best conditions for the system under consideration (removal factor of 2.69 and yield of 116%) were obtained at pH 8.0 using PEG molar mass of 8000 g mol(-1) and concentrations of PEG and citrate of 24 and 15%, respectively.
为降低用于疫苗制备的产气荚膜梭菌发酵液的毒性，我们开发了双水相体系以去除毒素激活蛋白酶。去除蛋白酶可抑制原毒素向活性毒素的转化。为确定发生相分离的条件，研究了由聚乙二醇（PEG）和柠檬酸钠形成的双节线在不同pH值和PEG摩尔质量下的情况。采用2(4)实验设计来评估PEG摩尔质量和浓度、柠檬酸盐浓度及pH值对蛋白酶分配系数、去除因子和蛋白酶去除率的影响。已发现PEG摩尔质量的同时增加和柠檬酸盐浓度的降低显著提高了去除因子，而蛋白酶去除率则呈现相反趋势。在所研究的体系中，最佳条件（去除因子为2.69，产率为116%）是在pH 8.0时获得的，此时PEG摩尔质量为8000 g mol(-1)，PEG和柠檬酸盐的浓度分别为24%和15%。
