一株产纤溶酶芽孢杆菌的鉴定及纤溶酶的分离纯化与性质分析

Identification of a fibrinolytic enzyme producing Bacillus pseudomycoides and purification and characterization of the enzyme

【目的】从假蕈状芽孢杆菌B-60菌株中纯化具有纤溶活性的单一组分,测定它的N-端氨基酸序列进行比对,并对单一组分的性质进行分析。【方法】利用纤维蛋白平板法检测纤溶酶活性,利用硫酸铵分级沉淀和阴离子交换色谱从假蕈状芽孢杆菌B-60菌株中纯化纤溶酶。【结果】从该菌株的发酵液中获得了一组纤溶酶单一组分(BpFE),它的表观分子量为34kDa。它在4℃～50℃活性较稳定,50℃以上活性急剧下降;作用最适pH值为pH5～6,在pH5～10活性较稳定,在pH3.0,活性几乎丧失;金属离子Ca2+、Mg2+、Mn2+对酶活有轻微促进作用,Cu2+则强烈抑制酶活。苯甲基磺酰氟(PMSF)完全抑制它的活性。BpFE经胰蛋白酶和胃蛋白酶降解后,活性上升。测得BpFEN-端15个氨基酸序列为VTGTNA VGTGKGVLG,序列比对结果表明,BpFEN-端15个氨基酸序列与来源于蜡状芽孢杆菌、苏云金芽孢杆菌、炭疽芽孢杆菌和乳杆菌的细菌裂解酶、中性蛋白酶、水解酶的部分序列同源性为100%。【结论】获得了一组纤溶酶单一组分,其N-端序列比对结果尚未发现与其同源性较高的纤溶酶,这为新型纤溶酶的开发提供了重要的理论依据。

[ Objective] To purify a single fibrinolytic enzyme from Bacillus pseudomycoides B-60 and to determine its N-terminal sequence and to characterize the fibrinolytic enzyme. [ Methods] We examined the fibrinolytic enzyme activity by fibrin plate and purified fibrinolytic enzyme by ammonium sulfate fractional precipitation and DEAE anion exchange chromatography. [ Results] Obtained a single protein fraction with fibrinolytic activity （BpFE） from B. pseudomycoides B-60. It appeared as a single band in the SDS-PAGE with a relative molecular weight of 34 kDa. The fibrinolytic activity of the protein was stable at 4 - 50℃ and at pH 5 - 10. The activity sharply decreased above 50℃, and the total loss of activity at pH 3.0. The enzymatic activity was slightly enhanced by the ions of Ca^2＋ , Mg^2＋ , Mn^2＋ , whereas strongly inhibited by Cu^2＋ ion. Phenylmethyl sulfonyl fluoride （PMSF） could completely inhibit its activity. In addition, the activity improved when the protein was enzymatically hydrolyzed using trypsin and pepsin. The first 15 amino acids of the N-terminal sequence of the enzyme were determined to be VTGTNAVGTGKGVLG. The partial amino acids sequence alignment study of the enzyme from B-60 strain with bacillolysin, neutral protease and hydrolase which were from B. cereus, B. thuringiensis, B. anthracis and Lactobacillus sp. was carried out, and there is a 100% homogeneity between them. [ Conclusion] We obtained a single fibrinolytic enzyme. Through its N-terminal sequence alignment study, a plasmin with higil homogeneity to this protein was not found yet. This provided a basis for further study of new thrombolytic drugs.