Pundir C S, Bhambi Manu, Chauhan Nar Singh
Biochemistry Research laboratory, Department of Biochemistry & Genetics, MD University, Rohtak 124001, Haryana, India.
Talanta. 2009 Mar 15;77(5):1688-93. doi: 10.1016/j.talanta.2008.10.004. Epub 2008 Oct 14.
Egg shell membrane is a novel, robust, microporous, cost effective, easily available organic support material. In recent studies egg shell membranes were utilized as organic support for enzyme immobilization. But low conjugation yield limits its application as good support for biotechnological industries. In present study egg shell membrane was chemically treated to introduce free functional groups for covalent linkage of proteins to increase its conjugation yield and stability of conjugate complex. Many enzymes were tested for immobilization on modified egg shell membrane like oxalate oxidase, glucose oxidase, peroxidase and lipase. A fifteen to sixteen fold increase in conjugation yield was observed when immobilization was performed after chemical treatment in comparison to immobilization on native membrane with slight change in specific activity of immobilized enzyme which ranges from 5% to 15%. Egg shell membrane bound enzymes showed slight changes in their kinetic properties after immobilization. Egg shell membrane bound oxalate oxidase shows detection limit of 1.5 microM when used for urinary oxalate determination. Egg shell membrane support shows no interference to enzyme activity and a good correlation of 0.99 was observed with the values estimated using commercially available Sigma kit. The immobilized oxalate oxidase, glucose oxidase, peroxidase and lipase were stable up to duration of 180 days and there is respective loss of 10%, 13%, 24%, and 33% of initial activity. Overall result strengthens our view of using chemically modified egg shell membrane as solid support for better immobilization of enzymes and can be used in various biotechnological applications.
蛋壳膜是一种新型、坚固、微孔、经济高效且易于获得的有机支撑材料。在最近的研究中，蛋壳膜被用作酶固定化的有机支撑物。但低结合产率限制了其作为生物技术产业良好支撑物的应用。在本研究中，对蛋壳膜进行化学处理以引入游离官能团，用于蛋白质的共价连接，以提高其结合产率和共轭复合物的稳定性。测试了多种酶在改性蛋壳膜上的固定化情况，如草酸氧化酶、葡萄糖氧化酶、过氧化物酶和脂肪酶。与在天然膜上固定化相比，化学处理后进行固定化时观察到结合产率提高了15至16倍，固定化酶的比活性略有变化，范围为5%至15%。蛋壳膜结合的酶在固定化后其动力学性质略有变化。用于尿草酸测定时，蛋壳膜结合的草酸氧化酶检测限为1.5微摩尔。蛋壳膜支撑物对酶活性无干扰，与使用市售Sigma试剂盒估计的值观察到良好的相关性，相关系数为0.99。固定化的草酸氧化酶、葡萄糖氧化酶、过氧化物酶和脂肪酶在长达180天的时间内保持稳定，初始活性分别损失10%、13%、24%和33%。总体结果强化了我们将化学改性蛋壳膜用作固体支撑物以更好地固定酶的观点，并且可用于各种生物技术应用。
