Yi Yunyu, Kermasha Selim, Neufeld Ronald
Department of Chemical Engineering, Queen's University, Kingston, Ont., Canada.
Biotechnol Bioeng. 2006 Dec 5;95(5):840-9. doi: 10.1002/bit.21027.
Immobilization of membrane proteins remains a challenge compared to soluble proteins. The membrane protein-chlorophyllase was successful entrapped in tetramethoxysilane (TMOS)-based sol-gel in the presence of lipid. Activity was examined against mixing rate, incubation temperature, time, substrate, acetone, and canola oil concentration. The external mass transfer of chlorophyll is not the rate-limiting step at higher mixing rates. Stability against temperature and acetone as denaturant was enhanced. In spite of the fact that an initial reaction lag phase was observed, 20% more chlorophyll was hydrolyzed, compared to reaction with free enzyme by the end of a 12 h assay. The initial lower activity demonstrated by entrapped chlorophyllase is likely due to the diffusion resistance of chlorophyll into and within the entrapment matrix. This hypothesis was substantiated by a low diffusion coefficient on the order of 10(-14) m(2)/s obtained for chlorophyll in nanoporous sol-gel particles. Pore size distribution of nanoporous wet TMOS-based sol-gel with or without protein was determined by thermoporometry. The change in pore morphology upon doping with chlorophyllase suggests that protein acts as a template during the sol-gel process.
与可溶性蛋白质相比，膜蛋白的固定化仍然是一个挑战。在脂质存在的情况下，膜蛋白叶绿素酶成功地被包埋在基于四甲氧基硅烷（TMOS）的溶胶 - 凝胶中。针对混合速率、孵育温度、时间、底物、丙酮和菜籽油浓度对活性进行了检测。在较高混合速率下，叶绿素的外部传质不是限速步骤。对温度和作为变性剂的丙酮的稳定性增强。尽管观察到初始反应滞后阶段，但在12小时测定结束时，与游离酶反应相比，水解的叶绿素多20%。包埋的叶绿素酶最初表现出较低的活性可能是由于叶绿素进入包埋基质以及在其中扩散的阻力。对于纳米多孔溶胶 - 凝胶颗粒中的叶绿素，获得了约10(-14) m(2)/s的低扩散系数，这证实了该假设。通过热孔率测定法确定了含或不含蛋白质的纳米多孔湿TMOS基溶胶 - 凝胶的孔径分布。用叶绿素酶掺杂后孔形态的变化表明蛋白质在溶胶 - 凝胶过程中充当模板。
