Caracterização bioquímica e molecular de enzimas trombolíticas obtidas de isolados microbianos dos Açores

Dissertação de Mestrado, Ciências Biomédicas, 13 de Maio de 2016, Universidade dos Açores.Bacillus sp. present a great diversity and a high productivity of protease. The group of Biotechnology Department of the Universidade dos Açores has a microbial isolates bank which includes about 1600 isolates of Bacillus sp. Some of the biggest potential thrombolytic enzymes are isolated from Bacillus sp. (streptokinase and natokinase). In this work we tested 79 isolates, previous tested to find a thrombolytic enzyme. The 79 isolates were tested for proteolytic activity in agar casein plate. Then the fibrinolytic activity and the thrombolytic activity of selected isolates were tested. Was determinate the group species of the bacterial isolates with higher activity and were selected 1 isolate from each group. The optimal pH and the optimal temperature of the extracellular protease produced by 11 isolates from Bacillus sp. were determinate also. A hemolytic test was made to the enzymes with a higher thrombolytic activity. After we check for fibrinolytic, thrombolytic and hemolytic activity were selected 2 isolates. These 2 isolates were tested for aPTT and PT activity, for plasminogen activator activity, fibrinogenolytic activity and euglobulin lysis time. 1 bacterial enzyme was selected for biochemical characterization and purification. From the 79 isolates with high proteolytic activity 27 were chosen by their high activity in pH 7.5 and 37˚C. From these 27 bacterial isolates 11 were spotted with fibrinolytic activity and thrombolytic activity (just 6% of the initial 79 isolates). 4 of these enzymes (S97B, S88A, S178C and 99D) belongs to Bacillus cereus group, 4 belongs to Bacillus mycoides group (S101C, S115C, S26A and S62A) and 3 belongs to Bacillus subtilis group (S157E, S122C and S150C). Were selected 2 isolates from the Bacillus mycoides group (S115C and S101C) because they didn’t present hemolytic activity. The isolate S115C didn’t interfere with aPPT test neither with PT test, otherwise S101C increase the aPPT, interfering with the normal coagulation time. Was also evidence that S101C is a plasminogen activator (t-PA) and S115C is not. Moreover, S115C presented more than 10x fibrinolytic activity then S101C, thus enzyme S115C was selected for biochemical characterization and purification. Biochemical characterization showed that S115C was a high inhibited by Benzamidine, STI, Chymostatin and PTCK indicating that S115C is a chymotrypsin-like serine protease. Suc-Ala-Ala- Pro-Phe-pNa was a specific substrate which indicates chymotrypsin activity. The activity of these enzyme was highly enhanced by Mn and slightly enhanced by Na and Ca2. This enzyme was inhibited by metal ions Mg2+, Cu+ and Ni2+. The zymogram of the purified enzyme revel a digestion band higher than 135 kDa and another band at 75kDa. SDS-page of purified fraction revel 2 bands one at 75 kDa and another at 140kDa. The SDS-page bands were cut and sent to analysis of mass spectrometry (Ms/Ms)