Balaraman K, Prabakaran G
Vector Control Research Centre (ICMR), Puducherry, India.
Indian J Med Res. 2007 Nov;126(5):459-64.
BACKGROUND & OBJECTIVE: Treatment of thromboembolic vascular disease has relied on anticoagulants. However, recognition that lysis of preformed fibrin could be accomplished in vivo by a process involving the conversion of inactive plasminogen to active plasmin enzyme led to an alternative enzyme-based approach. The drugs used for this therapy are called the fibrinolytic enzymes. In this study we attempted the production, purification and characterization of fibrinolytic enzyme from Bacillus sphaericus.
The seed was prepared in nutrient yeast salt medium (NYSM) in shake flask and organism was produced in 100 l pilot fermentor. Biomass was separated by centrifugation and crude protein was prepared by ammonium sulphate precipitation. Purification was done by ion exchange chromatography using Q sepharose followed by gel filtration chromatography using Sephacryl S- 300. Molecular weight was determined through HPLC. Fibrinolytic activity was assayed by fibrin plate method.
The production method yielded 64 mg/l of the crude enzyme and after purification it was 6.3 mg/l. The molecular weight of the compound was 18.6 kDa.
INTERPRETATION & CONCLUSION: The enzyme exhibited similar fibrinolytic activity as that of streptokinase, on fibrin plates that were devoid of plasminogen, suggesting that its fibrinolytic action is independent of plasminogen and it is not a plasminogen activator.
血栓栓塞性血管疾病的治疗一直依赖于抗凝剂。然而，认识到通过将无活性的纤溶酶原转化为活性纤溶酶的过程可以在体内完成预先形成的纤维蛋白的溶解，从而产生了一种基于酶的替代方法。用于这种治疗的药物被称为纤维蛋白溶解酶。在本研究中，我们尝试从球形芽孢杆菌中生产、纯化和表征纤维蛋白溶解酶。
在摇瓶中的营养酵母盐培养基（NYSM）中制备种子，并在100升中试发酵罐中培养该生物体。通过离心分离生物质，并通过硫酸铵沉淀制备粗蛋白。使用Q琼脂糖进行离子交换色谱，随后使用Sephacryl S - 300进行凝胶过滤色谱进行纯化。通过高效液相色谱法测定分子量。通过纤维蛋白平板法测定纤维蛋白溶解活性。
该生产方法产生了64毫克/升的粗酶，纯化后为6.3毫克/升。该化合物的分子量为18.6 kDa。
在不含纤溶酶原的纤维蛋白平板上，该酶表现出与链激酶相似的纤维蛋白溶解活性，表明其纤维蛋白溶解作用不依赖于纤溶酶原，且它不是纤溶酶原激活剂。
