Banca de DEFESA: STELIANE LIMA SANTOS

Uma banca de DEFESA de DOUTORADO foi cadastrada pelo programa.
STUDENT : STELIANE LIMA SANTOS
DATE: 28/11/2022
TIME: 14:00
LOCAL: PPGBA UFRPE - meet
TITLE:

Production, integrated purification of proteases with fibrinolytic activity by fungi of the genus Rhizopus and their biochemical characterization

KEY WORDS:

Rhizopus arrhizus, ATPS, Fibrinolytic protease, Purification

PAGES: 116
BIG AREA: Ciências Agrárias
AREA: Medicina Veterinária
SUMMARY:

Enzymes with fibrinolytic activity are obtained from different sources and can degrade fibrin, the main protein component of blood clots. The accumulation of fibrin in vessels can lead to thrombosis, a disease that occurs when there is an imbalance in the hemostatic system and plasmin, the enzyme responsible for breaking and removing clots, cannot dissolve them. The work in question has as its objectives to produce and purify proteases with fibrinolytic activity from filamentous fungi by submerged fermentation; carry out purification processes through an aqueous two-phase system (PEG/Citrate), in addition to biochemically characterize the fibrinolytic enzyme obtained from Rhizopus arrhizus UCP 1605. For production, a submerged fermentation was carried out with fungi of the genus Rhizophus. Then a complete 2³ factorial design to determine the best conditions for cultivation of the selected microorganism, for this, the influence of the variables substrate type (TS), substrate concentration (CS) and glucose concentration (GC) were evaluated under the production of fibrinolytic proteases, resulting in the best production condition (3% soybean MS-2 medium, and 0.5% glucose, submitted to 30ºC under stirring at 120 rpm for 96 hours of fermentation). The enzyme produced was partially purified by aqueous two-phase systems (SDFA) consisting of polyethylene glycol (PEG) and sodium citrate, being carried out according to a factorial design 2⁴, to evaluate the influence of the independent variables: molar mass of PEG (MPEG), PEG concentration (CPEG), sodium citrate concentration (Ccit) and pH on the partition coefficient (K), yield (Y%) and purification factor (PF). Then the enzyme was characterized in kinetic biochemical parameters. A 2⁴ factorial design was also carried out to evaluate the influence of MPEG, CPEG, Ccit and pH on K and Y in extractive fermentation. The fibrinolytic protease from Rhizopus arrhizus UCP 1605 was produced under conditions of 3% soybean and 0.5% glucose, under agitation of 120 rpm, at 30ºC for 96 hours of submerged fermentation, presenting a protease activity of 10.37 U /mL and a fibrinolytic activity with a halo of 31 mm, corresponding to an activity of 850.60 U/mL. As for the extraction in SDFA, the best results were obtained in the assay formed by PEG 8000 g/mol 24.0 % (m/m), 15 % (m/m) of sodium citrate, and pH 8. In this condition, the enzyme partitioned preferably for the PEG-rich phase, with a K of 1.58, FP 4.07, Y of 97.0% and a fibrinolytic activity with a halo of 16 mm, corresponding to an activity of 44.39 U/mL. In the extractive fermentation process, the enzyme partitioned for both phases, having the best condition in the test composed of PEG 8000 g/mol, at a concentration of 20%, 15% of citrate and pH 8.0, presenting a K of 2.4 activity recovery (Y) = 71.5% and 3.83 U/mL of protease activity. The enzyme presents in the enzymatic extract and in the SDFA presented an optimal temperature of 50ºC and an optimal pH of 8. As for the kinetic parameters, the enzyme presented a Michaelis-Menten constant (Km) of 4.06 mg/mL with a maximum velocity of 45.05 U/mL. These results demonstrate the potential of the production of Rhizopus arrhizus UCP 1605 in submerged fermentation and of the fibrinolytic proteases extraction processes and their possibility to be explored in industrial applications as candidates for thrombolytic agents.

BANKING MEMBERS:
Presidente - TATIANA SOUZA PORTO
Externa à Instituição - AMANDA EMMANUELLE SALES CONNIFF
Externa à Instituição - CAMILA SOUZA PORTO - UFAL
Externa à Instituição - MARLLYN MARQUES DA SILVA - UFRPE
Externo à Instituição - THIAGO PAJEÚ NASCIMENTO - UFPI