Department of Morphology and Animal Physiology, Federal Rural University of Pernambuco, 52171-900 Recife, PE, Brazil.
Appl Biochem Biotechnol. 2013 Aug;170(7):1676-88. doi: 10.1007/s12010-013-0306-z. Epub 2013 May 29.
Fibrinolytic proteases are enzymes that degrade fibrin; these enzymes are a promising alternative for thrombolytic therapy, and microorganisms produce them. The aim of this study was to evaluate the optimum conditions for the integrated production and purification of fibrinolytic protease from Bacillus sp. UFPEDA 485. Extractive fermentation was carried out in a culture medium containing soybean flour and by adding polyethylene glycol (PEG) and Na2SO4 according to a 2(3) experimental design. In all assays, the enzyme preferentially partitioned to the bottom phase (K < 1), with an optimum activity of 835 U ml(-1) in the bottom phase (salt-rich phase). The best conditions for extractive fermentation were obtained with 18 % PEG 8000 and 13 % Na2SO4. Characterization showed that it is a metalloprotease, as a strong inhibition-residual activity of 3.13 %-occurred in the presence of ethylenediaminetetraacetic acid. It was also observed that enzymatic activity was stimulated in the presence of ions: CaCl2 (440 %), MgCl2 (440 %), FeSO4 (268 %), and KCl (268 %). The obtained results indicate that the use of a low-cost substrate and the integration of fermentation with an aqueous two-phase system extraction may be an interesting alternative for the production of fibrinolytic protease.
纤维蛋白溶酶是降解纤维蛋白的酶；这些酶是溶栓治疗的一种有前途的替代方法，微生物可以产生它们。本研究旨在评估从芽孢杆菌 UFPEDA 485 中综合生产和纯化纤维蛋白溶酶的最佳条件。在含有大豆粉的培养基中进行萃取发酵，并根据 2(3)实验设计添加聚乙二醇(PEG)和 Na2SO4。在所有试验中，酶优先分配到底相(K<1)，底相(盐富集相)的最佳活性为 835 U ml(-1)。萃取发酵的最佳条件为 18%PEG 8000 和 13%Na2SO4。特性表明它是一种金属蛋白酶，因为在存在乙二胺四乙酸的情况下，抑制残留活性强达 3.13%。还观察到酶活性在存在离子时受到刺激：CaCl2(440%)、MgCl2(440%)、FeSO4(268%)和 KCl(268%)。所得结果表明，使用低成本的底物并将发酵与双水相萃取相结合可能是生产纤维蛋白溶酶的一种有趣的替代方法。
