Departamento de Biología Medioambiental, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.
Bioresour Technol. 2012 Jul;115:177-82. doi: 10.1016/j.biortech.2011.09.035. Epub 2011 Sep 16.
Cross-linked enzyme aggregates of poly-3-hydroxybutyrate (PHB) depolymerase from Streptomyces exfoliatus (PhaZ(Sex)-CLEAs) have been prepared. Acetone was used as the precipitating agent, while addition of bovine serum albumin (BSA) facilitated CLEAs formation. Conditions for enzyme precipitation and cross-linking have been optimized, and confocal scanning microscopy showed a homogeneous enzyme distribution in the biocatalyst. Obtained PhaZ(Sex)-CLEAs presented an average size of 50-300 μm, showing a high PHB depolymerase activity of 255 U/g wet biocatalyst at 40°C and pH 7.0. Temperature-activity profile of PhaZ(Sex)-CLEAs at pH 8.0 showed that the highest activity for pNPB hydrolysis was achieved at 60°C, whereas pH-activity profile at 40°C indicated that highest activity for PHB hydrolysis was achieved at pH 7.0. Additionally, immobilized biocatalyst could be recycled at least for 20 consecutive batch reactions without loss of catalytic activity, and showed higher pH and temperature stability, and better tolerance to several organic solvents than its soluble counterpart.
已制备了来自腐烂链霉菌（PhaZ(Sex)-CLEAs）的聚 3-羟基丁酸酯（PHB）解聚酶的交联酶聚集体（CLEAs）。使用丙酮作为沉淀剂，而添加牛血清白蛋白（BSA）有助于 CLEAs 的形成。已经优化了酶沉淀和交联的条件，共焦扫描显微镜显示在生物催化剂中存在均匀的酶分布。获得的 PhaZ(Sex)-CLEAs 的平均粒径为 50-300μm，在 40°C 和 pH 7.0 下表现出 255U/g 湿生物催化剂的高 PHB 解聚酶活性。在 pH 8.0 下的 PhaZ(Sex)-CLEAs 的温度-活性曲线表明，用于 pNPB 水解的最高活性在 60°C 下实现，而在 40°C 下的 pH-活性曲线表明，用于 PHB 水解的最高活性在 pH 7.0 下实现。此外，固定化生物催化剂在没有失去催化活性的情况下至少可以循环使用 20 次连续批次反应，并且显示出比其可溶性对应物更高的 pH 和温度稳定性，以及对几种有机溶剂的更好耐受性。
