CELLULASE IMMOBILIZATION ON POLY(METHYL METHACRYLATE) NANOPARTICLES BY MINIEMULSION POLYMERIZATION

Abstract Cellulases are efficient enzymes for the conversion of cellulose into glucose. Their use in immobilized form enables them to be reused in successive cycles in many biotechnological processes. Unlike conventional methods of immobilization by covalent bonding, in miniemulsion polymerization the immobilization of enzyme and the synthesis of polymer nanoparticles (support) occur simultaneously. Based on these aspects, the immobilization of cellulose on poly(methyl methacrylate) (PMMA) nanoparticles by miniemulsion polymerization was studied. The surfactant type (non-ionic and ionic) and latex pH showed great influence on cellulase activity. High activity values were obtained only when non-ionic surfactant (Lutensol AT50) and buffering agent (NaHCO3) were used simultaneously. MMA polymerization rate and final monomer conversion were not affected by the presence of cellulase. The maximum immobilization efficiency (60%) was obtained when 6 wt.% of cellulase was used and stable PMMA nanoparticles (133 nm) were obtained. The relative activity profile of immobilized cellulase, for pH as well as temperature, was similar to that reported for the free form. Immobilized enzyme keeps its activity throughout seven days when stored at 4 ºC and phosphate buffer pH 6.0. Based on the results obtained in this work, miniemulsion polymerization as a method for cellulase immobilization on PMMA nanoparticles showed to be a promising technique with high possibility of industrial application.

摘要 纤维素酶（cellulase）是将纤维素（cellulose）转化为葡萄糖（glucose）的高效酶制剂，以固定化形式使用可使其在诸多生物技术工艺的循环流程中重复利用。与传统共价结合（covalent bonding）固定化方法不同，细乳液聚合（miniemulsion polymerization）过程中酶的固定化与聚合物纳米颗粒（载体）的合成可同步完成，基于此本研究探讨了通过细乳液聚合法将纤维素酶固定于聚甲基丙烯酸甲酯（poly(methyl methacrylate), PMMA）纳米颗粒上的工艺。研究表明，表面活性剂（surfactant）类型（非离子型与离子型）及胶乳（latex）pH值对纤维素酶活性影响显著，仅当同时使用非离子型表面活性剂Lutensol AT50与缓冲剂（buffering agent）碳酸氢钠（NaHCO₃）时方可获得较高酶活性；甲基丙烯酸甲酯（MMA）的聚合速率及最终单体转化率不受纤维素酶存在的影响，当添加6 wt.%的纤维素酶时可实现最高60%的固定化效率（immobilization efficiency），同时得到粒径稳定为133 nm的PMMA纳米颗粒；固定化纤维素酶的pH及温度相对活性（relative activity）曲线与已报道的游离形式（free form）纤维素酶较为相似，在4℃、pH 6.0的磷酸盐缓冲液中储存时，固定化酶的活性可维持7天。基于本研究所得结果，细乳液聚合法作为将纤维素酶固定于PMMA纳米颗粒上的手段，展现出良好的应用前景与较高的工业转化潜力。