Graduate Program in Chemical Engineering, Department of Chemical Engineering, Federal University of São Carlos, Rod. Washington Luís, km 235, SP-310, São Carlos 13565-905, Brazil.
Departmento de Biocatálisis, ICP-CSIC, Campus UAM-CSIC Madrid, 28049 Madrid, Spain.
Molecules. 2018 Nov 16;23(11):2993. doi: 10.3390/molecules23112993.
The preparation of highly porous magnetic crosslinked aggregates (pm-CLEA) of porcine pancreas lipase (PPL) is reported. Some strategies to improve the volumetric activity of the immobilized biocatalyst were evaluated, such as treatment of PPL with enzyme surface-modifying agents (polyethyleneimine or dodecyl aldehyde), co-aggregation with protein co-feeders (bovine serum albumin and/or soy protein), use of silica magnetic nanoparticles functionalized with amino groups (SMNPs) as separation aid, and starch as pore-making agent. The combination of enzyme surface modification with dodecyl aldehyde, co-aggregation with SMNPs and soy protein, in the presence of 0.8% starch (followed by hydrolysis of the starch with α-amylase), yielded CLEAs expressing high activity (immobilization yield around 100% and recovered activity around 80%), high effectiveness factor (approximately 65% of the equivalent free enzyme activity) and high stability at 40 °C and pH 8.0, i.e., PPL CLEAs co-aggregated with SMNPs/bovine serum albumin or SMNPs/soy protein retained 80% and 50% activity after 10 h incubation, respectively, while free PPL was fully inactivated after 2 h. Besides, highly porous magnetic CLEAs co-aggregated with soy protein and magnetic nanoparticles (pm-SP-CLEAs) showed good performance and reusability in the hydrolysis of tributyrin for five 4h-batches.
报告了一种猪胰腺脂肪酶（PPL）的高多孔磁性交联聚集物（pm-CLEA）的制备方法。评估了一些提高固定化生物催化剂体积活性的策略，例如用酶表面改性剂（聚乙烯亚胺或十二醛）处理 PPL、与蛋白质共饲料（牛血清白蛋白和/或大豆蛋白）共聚聚集、使用功能化有氨基的硅胶磁性纳米颗粒（SMNPs）作为分离助剂，以及使用淀粉作为造孔剂。酶表面修饰与十二醛、SMNPs 和大豆蛋白共聚聚集相结合，在 0.8%淀粉存在下（随后用α-淀粉酶水解淀粉），产生了表达高活性（固定化产率约为 100%，回收活性约为 80%）、高有效因子（约为游离酶活性的 65%）和在 40°C 和 pH8.0 下高稳定性的 CLEAs，即与 SMNPs/牛血清白蛋白或 SMNPs/大豆蛋白共聚聚集的 PPL CLEAs 在孵育 10 小时后分别保留 80%和 50%的活性，而游离 PPL 在 2 小时后完全失活。此外，与大豆蛋白和磁性纳米颗粒共聚聚集的高多孔磁性 CLEAs（pm-SP-CLEAs）在五个 4 小时批次的三丁酸甘油酯水解中表现出良好的性能和可重复使用性。
