Fibrinolytic enzymes production by Bionectria sp. LY 4.1: the relevance of inoculum homogeneization and pH-controlled conditions at fermenter scale

CARO F.C.; BABOT J.D.; DELGADO O.D.; FARIÑA J.I.

Congreso; XI Congreso Argentino de Microbiología General SAMIGE; 2015

Fibrin accumulation in blood vessels usually results in thrombosis, which can thereafter lead to myocardial infarction and other cardiovascular diseases. Fibrin is the primary protein component of blood clots and is physiologically formed from fibrinogen by the catalytic action of thrombin. During fibrinolysis, the insoluble fibrin fiber is hydrolyzed into fibrin degradation products by plasmin. Nowadays, fibrinolytic enzymes of microbial origin have attracted more attention than typical thrombolytic agents used for thrombolytic therapy. This choice is based on the high price and the undesirable side effects of the latter. The aim of this work was to study the production of fibrinolytic enzymes by Bionectria sp. LY 4.1, a wild fungus isolated from Las Yungas rainforest and already described in our group. In this case, we focused on the fermentation process upstream optimization in order to increase fibrinolytic enzymes production at fermenter scale. At first, inoculation process was standardized by hand-blender-aided homogenization of mycelial suspensions and evaluating the influence of power input and the number of pulses. The production of fibrinolytic enzymes was also preliminary evaluated at different initial cultivation pHs, from 4 to 8, at shake-flask scale and by using an 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 by controlling culture broth pH (with 1 N NaOH) at the optimal value obtained in previous assays, and results were comparatively assessed. Fibrinolytic activity was determined by the fibrin plate test and by 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 fibrinolytic activity (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 a 20% higher production was obtained at pH=8 and Accordingly, this value was selected for testing at fermenter scale. Batch fermentations were comparatively run under free pH (firstly set in culture medium at 6.6 and left uncontrolled afterwards) and with automatic control at pH=8. Further operative conditions 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/ml under controlled pH. These findings provide first clues into the possibilities for the upstream fermentation process optimization through the automatic pH controlling strategy. Following studies will be focused on the use of a different pH controlling agent and further operative conditions. The elucidation of these optimal parameters will then be useful for the subsequent scaling-up.