Abstract:

Silica was modified by a polyethyleneimine (PEI)/dopamine (DA) co-deposition method, and then carbonic anhydrase (CA) was covalently immobilized via glutaraldehyde (GA). First, the effects of the mass ratio of PEI and DA, deposition time on the deposition rate were investigated, and silica microspheres before and after modification were characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM). Then, the influences of the immobilization conditions including the deposition rate, the addition amount of PEI/PDA-SiO₂, CA and GA concentration on the activity recovery of immobilized enzyme were studied. At last, the storage stability and reusability of immobilized enzyme were also examined. The results show that the deposition rate increased firstly and then decreased with the mass ratio of PEI and DA increased, and reached the maximum at a mass ratio of 1∶1. The deposition rate increased linearly with the time prolonged, which respectively reached 1.33% and 0.5% after 10 h for PEI/DA co-deposited system and pure DA-deposited that. The deposition rate had no obvious effect on the content of N element and activity recovery of immobilized CA. For enzyme immobilization, there was a saturated addition amount of the carrier which was 0.25 g PEI/PDA-SiO₂/mg CA, and the optimal concentration of CA and GA was 0.8 mg/ml and 0.1%(mass), respectively. Under these conditions, the activity recovery of CA-PEI/PDA-SiO₂ was about 78.8%. After 30 days of storage at 25℃, the remaining activity of immobilized CA was 77.2% while that of free CA was only 12%. The relative activity of immobilized CA was 88.3% after undergoing 10 cycles.

Key words: polyethylenimine, dopamine, silica, enzyme, immobilization, biotechnology

摘要：

利用聚乙烯亚胺（PEI）/多巴胺（DA）共沉积法改性氧化硅，并以此为载体固定化碳酸酐酶（CA）。考察了PEI/DA质量比、沉积时间对沉积率的影响，用傅里叶红外光谱（FTIR）和扫描电子显微镜（SEM）对改性前后的微球进行了表征；研究了沉积率、载体用量、酶浓度及戊二醛（GA）浓度对固定化酶活回收率的影响；考察了固定化酶的储存稳定性和重复利用性。结果表明，随PEI/DA质量比增加，沉积率先增加后降低，质量比为1∶1时最大；随沉积时间增加，沉积率线性增长，10 h后PEI/DA体系沉积率为单独DA沉积改性的2.66倍，但沉积时间对N元素含量和酶活回收率影响不大；酶固定化时载体用量存在饱和值，CA和GA浓度的最优值分别为0.8 mg/ml和0.1%(质量)，此时酶活回收率可达78.8%。在25℃下储存30 d后，固定化酶的保留活性为77.2%，而游离酶只有12%；重复使用10次后，固定化酶仍能保持88.3%的相对活性。

关键词: 聚乙烯亚胺, 多巴胺, 二氧化硅, 酶, 固定化, 生物技术