药用级微生物谷氨酰胺转胺酶的纯化工艺研究

Large Scale Purification of Pharmaceutical Grade Microbial Transglutaminase from Streptoverticillium mobaraense

为了提高谷氨酰胺转胺酶的纯度和扩展在医药领域的应用,探索了一种适合工业化生产的、安全高效的微生物谷氨酰胺转胺酶纯化方法。轮枝链霉菌发酵后,经离心10 000 r/min 4℃除去菌体,调节发酵液电导率至4.1mS/cm和pH6.0后,以直线流速60cm/h通过SP Sepharose FF阳离子交换层析柱对目的蛋白高选择性和高载量地捕获,再通过phenyl sepharose 6 FF(high sub)疏水层析柱进行精细纯化。纯化后经SDS-PAGE鉴定纯度达到95%以上,HPLC分析纯度>99%。鲎试剂测定内毒素含量为0.013EU/ml,达到中国药典中血制品要求的低于0.15EU/ml标准。

A safe efficient microbial transglutaminase purification method applied for industrial manufacture was introduced in order to improve the purity of transglutaminase and extend its applications for pharmaceuticals. Cultures was centrifuged at 10 000 r/min 4℃ to remove the cells before adjusting conductivity to 4.1 mS/cm and pH to 5.5 for purification. The sample was purified by cation-exchange chromatography at a linear velocity of 60 cm/h in the first step. In this step SP Sepharose FF as a packing provided high selectivity and high loading for the target protein. After primary purification, the sample was further processed/isolated with hydrophobic chromatography packed with phenyl Sepharose 6 FF （ high sub）. SDS-PAGE pattern manifested a purity of over 95% and HPLC analysis indicated a purity of above 99% were achieved after this two purification step method. LPS of the purified sample was determined at 0. 013 EU/ml with tachypleus amebocyte lysate, met the blood products requirement （ 〈 0. 15 EU/ml ） provided in Chinese Pharmacopoeia. Thus the method described/ established is efficient and practical in microbial transglutaminase purification.