Proteolytic Activity Assays in Haloarchaea

Paggi, Roberto A.; Giménez, María Inés; Castro, Rosana Esther de

doi:10.1007/978-1-0716-2445-6_20

Cited by 2 publications

(1 citation statement)

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“…Protease activity was determined using a previously described method with minor modifications [ 15 ]. The mixture containing 50 μL azocasein (2% solution in 10 mM Tris-HCl buffer pH 8.0) and samples (100 μL) were incubated at 40 °C for 60 min and then stopped with 50 μL of trichloroacetic acid (10%).…”

Section: Methodsmentioning

confidence: 99%

Partial Purification and Biochemical Evaluation of Protease Fraction (MA-1) from Mycoleptodonoides aitchisonii and Its Fibrinolytic Effect

et al. 2023

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The antioxidative proteolytic fraction, MA-1, was partially purified from Mycoleptodonoides aitchisonii. MA-1 was purified to homogeneity using a two-step procedure, which resulted in an 89-fold increase in specific activity and 42.5% recovery. SDS-PAGE revealed two proteins with a molecular weight of 48 kDa. The zymography results revealed proteolytic activity based on the MA-1 band. MA-1 was found to be stable in the presence of Na+, Ca2+, Fe3+, K+, and Mg2+. MA-1 was also stable in methanol, ethanol, and acetone, and its enzyme activity increased by 15% in SDS. MA-1 was inhibited by ethylenediaminetetra-acetic acid or ethylene glycol tetraacetic acid and exerted the highest specificity for the substrate, MeO-Suc-Arg-Pro-Tyr-pNA, for chymotrypsin. Accordingly, MA-1 belongs to the family of chymotrypsin-like metalloproteins. The optimum temperature was 40 °C and stability was stable in the range of 20 to 35 °C. The optimum pH and stability were pH 5.5 and pH 4–11. MA-1 exhibited stronger fibrinolytic activity than plasmin. MA-1 hydrolyzed the Aα, Bβ, and γ chains of fibrinogen within 2 h. MA-1 exhibited an antithrombotic effect in animal models. MA-1 was devoid of hemorrhagic activity at a dose of 80,000 U/kg. Overall, our results show that M. aitchisonii produces an acid-tolerant and antioxidative chymotrypsin-like fibrinolytic enzyme, and M. aitchisonii containing MA-1 could be a beneficial functional material for the prevention of cardiovascular diseases and possible complications.

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Section: Methodsmentioning

confidence: 99%