Production and Characterization of New Fibrinolytic Protease from <i>Mucor subtillissimus</i> UCP 1262 in Solid-State Fermentation

Abstract

Fibrinolytic enzymes have received attention regarding their medicinal potential for thrombolytic diseases, a leading cause of morbidity and mortality worldwide. Various natural enzymes purified from animal, plant and microbial sources have been extensively studied. The aim of this work was to produce fibrinolytic protease by solid state fermentation using agro industrial substrates. Rhizopus arrhizus var. arrhizus UCP 1295 and Mucor subtillissimus UCP 1262 filamentous fungi species isolated from soil of Caatinga-PE, Brasil, were used as producer microorganisms. Wheat bran was shown to be the best substrate for the production of the enzyme and by using a 2 3 full factorial design the main effects and interactions of the quantity of the substrate wheat bran, moisture and temperature on the fibrinolytic enzyme production and protease were evaluated. The best results for fibrinolytic and protease activities, 144.58 U/mL and 48.33 U/mL, respectively, were obtained with Mucor subtillissimus UCP 1262 using as culture medium 3 g wheat bran, 50% moisture at a temperature of 25˚C for 72 hours. The optimum temperature for the produced enzyme was 45˚C and most of its original activity was retained after being subjected to 80˚C for 120 min. The pro-* Corresponding author.T. P. Nascimento et al. 82tease activity was enhanced by K + , Ca + and Mn + ; but with Cu + there was an inhibition. The specificity to chromogenic substrate and the inhibition by PMSF indicates that it is a chymotrypsin-like serine protease. Presented results suggest that this enzyme produced by solid-state fermentation is an interesting alternative as a candidate for thrombolytic therapy.

Purification of a fibrinolytic protease from Mucor subtilissimus UCP 1262 by aqueous two-phase systems (PEG/sulfate)

“…125 mL-Erlenmeyer flasks containing 3.0 g of wheat bran as a substrate (moisture content of 50%) were sterilized by autoclaving at 121 • C, 1 atm for 20 min, inoculated with the above suspension of M. subtilissimus UCP 1262 spores, and incubated at 25 • C for 72 h. Both the microorganism and the substrate were chosen based on previous work [23].…”

“…Enzyme extraction was performed after 72 h of fermentation. For this purpose, 7.5 mL of 245 mM sodium phosphate buffer, pH 7, were added per g of substrate, and the flasks were placed in an orbital shaker at 150 rpm for 90 min at room temperature, as previously described [23]. Samples were then centrifuged at 3500 rpm for 10 min, and the supernatant was used for determination of protease and fibrinolytic activities.…”

Agro-industrial wastes and their utilization using solid state fermentation: a review

“…SSF has been mainly employed from ancient time for processing of foods, but nowadays it is gaining a lot of attention due to the increasing use of different types of organic wastes and the larger production of value-added products (Pandey et al 2000;Wang and Yang 2007). The search for sustainable and green processes for bioconversion of organic wastes into valuable products could substitute Aspergillus oryzae Soybean meal (waste) Thakur et al (2015) Rhizopus arrhizus and Mucor subtillissimus Caorncob cassava peel, soybeans, wheat bran, and citrus pulp Nascimento et al (2015) Aspergillus niger Rice bran, wheat bran, black gram bran, GOC, and COC Mahalakshmi and Jayalakshmi (2016) Aspergillus terreus Palm oil cake Rahman et al (2016) non-renewable materials and also transform chemical processes into cleaner practices in the industrial sector that highlights the potential of SSF. The particular interest of SSF is due to its relatively simple process that uses abundant low-cost biomaterials with minimal or no pretreatment for bioconversion, less waste water generation, and the capacity for simulating similar micro-environments, favorable to microorganism growth (Singhania et al 2009).…”

The Application of Solid State Fermentation for Obtaining Substances Useful in Healthcare

“…Once the main substrate is prepared, there are other experimental conditions that need to be optimised. In cited papers the major factor contributing to obtained results was moisture which was at least 50% [69,110]. The next crucial factor was medium composition.…”