黏细菌抗凝溶栓双功能蛋白MF-1的纯化及其酶学性质

对黏细菌Angiococcus sp.的抗凝溶栓双活性蛋白MF-1进行纯化、鉴定并对其酶学性质进行初步研究。采用丙酮分级沉淀法、DEAE-Sepharose离子交换层析和Sephadex G-50分子筛层析对发酵液进行纯化,用SDS-PAGE和等电点聚焦电泳对其进行鉴定,并用纤维蛋白平板法和水解酪蛋白法对其酶学性质进行检测。结果表明：经过一系列的纯化步骤分离得到该蛋白相对分子质量为3.2×10^4,等电点为8.5,酶的比活力为30761.57U/mg,活性回收率为13.9%;溶栓活性的最适反应温度为35℃,最适反应pH为8.0;抗凝时间大于10min,且酶活性十分稳定,在35℃下保温72h后仍有89%活性。首次从黏细菌中分离得到具有较高抗凝和溶栓双活性的MF-1蛋白,且稳定不易失活,具有开发成为创新溶栓药物的潜力。

Purification and characterization of protein MF-1 with fibrinolytic and anticoagulant activities from myxobacteria

Protein MF-1 produced by fermentation with myxobacterium Angiococcus sp.was purifed and identified,the fibrinolytic and anticoagulant activities were assayed. Preliminary enzymatic properties of the protein were investigated.The protein in the fermentation broth was precipitated with acetone, purified by DEAE-Sepharose and Sephadex G-50 chromatography,identified by SDS-PAGE and isoelectric focusing electrophoresis, and assayed by the fibrin plate and the casein hydrolysate.The results showed that the molecular weight of the protein was 3.2×10~4.PI was 8.5.The fibrinolytic activity reached 30 761.57 U/mg.The recovery activity rate was 13.9%.the optimal temperture of the fibrinolytic activity was 35℃,pH=8.0.and the activity remained 89% after keeping at 35 ℃ for 72 h.The anticoagulant time was more than 10 min. It was the first report of MF-1 from myxobacterium with both fibrinolytic and anticoagulant activities.