模板法制备多孔交联脂肪酶聚集体碳酸钙及其特性

Preparation and properties of porous crosslinked lipase polymeric by calcium carbonate template method

针对传统交联脂肪酶聚集体(cross-linked lipase aggregates,CLEAs)表面密实、比表面积小、无太多孔隙结构、在催化过程中存在扩散限制、影响酶催化效率等问题,该文通过脂肪酶、氯化钙、碳酸钠、硫酸铵共沉淀制备脂肪酶/碳酸钙微球,再加入二硫苏糖醇(dithiothreitol,DTT)进行脂肪酶交联自组装,然后用乙二胺四乙酸二钠(EDTA)去除碳酸钙模板剂,制得多孔交联脂肪酶聚集体微球(p-CLEAs),对其制备条件、结构特征、酶学性质进行研究。结果表明,最佳制备条件为:Ca^2+浓度0.35 mol/L、脂肪酶与Ca^2+比例5:1、沉淀剂饱和度80%、沉淀pH值为8、沉淀时间45 min、DTT体积分数0.2%、交联时间40 min。与常规CLEAs相比,所制备的p-CLEAs在甲醇耐受性、热稳定性和pH值稳定性方面均有明显改善,4℃保藏6个月,仍保持较高的活性。其结构稳定,形貌、孔道尺寸可调控,呈多孔结构,这种多孔结构使得底物分子更容易进入脂肪酶的活性位点,不仅降低了传质限制,还提高了催化效率,具有较高的催化活性。

In view of the problems such as compact surface,small specific surface area and lack of much pore structure of traditional cross-linked lipase aggregates(CLEAs),which further affects the catalytic efficiency of the enzyme in the catalytic process.To solve above problems,this study prepared porous-cross linked enzyme aggregates(p-CLEAs)were synthesized by in-situ co-precipitation method using CaCO3 microparticles as templates.The preparation procedure involves crude lipase was immobilized as CLEAs via precipitation with ammonium sulfate and entrapping this lipase molecules into CaCO3 templates,followed by DTT(dithiothreitol)induced assemble of lipase molecules to from lipase microparticles(Lipase molecules were assembled into a microparticle by the internal using disulfide bonds within lipase molecules as molecular linkers and stimulated by dithiothreitol)and finally the removal of CaCO3 templates by EDTA to form pores in the CLEAs.The preparation conditions,structural characteristics and enzymatic properties were studied.The results showed that the best preparation conditions were:mass concentration of concentration of Ca^2+ 0.35 mol/L,the ratio of lipase to Ca^2+ is 5:1,saturation of precipitator is 80%,precipitation pH value is 8,precipitation time is 40 min,DTT volume fraction is 0.2%,and crosslinking time is 40 min,Compared with conventional CLEAs,the prepared p-CLEAs presented a porous structure and showed significant improvement in methanol tolerance,thermal stability and pH value stability.It was preserved at 4℃ for 6 months and still maintained a high activity.This porous structure makes it easier for substrate molecules to enter the active site of lipase,which not only reduces the mass transfer limitation,but also improves the catalytic efficiency.Therefore,p-CLEAs lipase has a high catalytic activity.