CONICET | Buscador de Institutos y Recursos Humanos

Fibrin accumulation in blood vessels usually results in thrombosis, which can thereafter lead tomyocardial infarction and other cardiovascular diseases. Fibrin is the primary protein component ofblood clots and is physiologically formed from fibrinogen by the catalytic action of thrombin. Duringfibrinolysis, the insoluble fibrin fiber is hydrolyzed into fibrin degradation products by plasmin.Nowadays, fibrinolytic enzymes of microbial origin have attracted more attention than typicalthrombolytic agents used for thrombolytic therapy. This choice is based on the high price and theundesirable side effects of the latter. The aim of this work was to study the production of fibrinolyticenzymes by Bionectria sp. LY 4.1, a wild fungus isolated from Las Yungas rainforest and alreadydescribed in our group. In this case, we focused on the fermentation process upstream optimization inorder to increase fibrinolytic enzymes production at fermenter scale. At first, inoculation process wasstandardized by hand-blender-aided homogenization of mycelial suspensions and evaluating theinfluence of power input and the number of pulses. The production of fibrinolytic enzymes was alsopreliminary evaluated at different initial cultivation pHs, from 4 to 8, at shake-flask scale and by usingan optimized production medium based on glucose, soy peptone, NaCl and MgSO4. Subsequently,batch cultures were carried out with a 1-L working volume fermenter either at uncontrolled pH or bycontrolling culture broth pH (with 1 N NaOH) at the optimal value obtained in previous assays, andresults were comparatively assessed. Fibrinolytic activity was determined by the fibrin plate test andby using a plasmin standard curve. Inoculum standardization showed that, at 48 h of fermentation,inoculum homogenized with a higher number of pulses allowed to obtain an increased fibrinolyticactivity (495 U plasmin/ml) as compared to the process started with a slightly homogenized inoculum(170 U plasmin/ml). The screening for optimum cultivation pH in shake-flasks assays revealed that a20% higher production was obtained at pH=8 and accordingly, this value was selected for testing atfermenter scale. Batch fermentations were comparatively run under free pH (firstly set in culturemedium at 6.6 and left uncontrolled afterwards) and with automatic control at pH=8. Further operativeconditions were set as follows: agitation, 200 rpm; temperature, 25°C and airflow rate, 1.5 vvm.Fibrinolytic enzymes titers reached 1888 U plasmin/ml at pH-free conditions vs. 2437 U plasmin/mlunder controlled pH. These findings provide first clues into the possibilities for the upstreamfermentation process optimization through the automatic pH controlling strategy. Following studies willbe focused on the use of a different pH controlling agent and further operative conditions. Theelucidation of these optimal parameters will then be useful for the subsequent scaling-up.

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