Banca de QUALIFICAÇÃO: LÍGIA MARIA GONÇALVES FERNANDES

Uma banca de QUALIFICAÇÃO de DOUTORADO foi cadastrada pelo programa.
STUDENT : LÍGIA MARIA GONÇALVES FERNANDES
DATE: 21/12/2023
TIME: 09:00
LOCAL: PPGBA UFRPE - meet
TITLE:

DEVELOPMENT OF BIOPOLIMER HYDROGEL CONTAINING PROTEASES WITH COLLAGENOLYTIC ACTIVITY FROM Aspergillus heteromorphus URM 0269 FOR POTENTIAL USE IN WOUND HEALING

KEY WORDS:

Aspergillus heteromorphus, Protease with collagenolytic activity, Solid state fermentation, Submerged fermentation, Bioreactor, Ion exchange chromatography, Biochemical characterization, Kinetics and thermodynamics

PAGES: 104
BIG AREA: Ciências Agrárias
AREA: Medicina Veterinária
SUMMARY:

This study aimed to produce a protease with collagenolytic activity from the filamentous fungus Aspergillus heteromorphus URM 0269, using substrates such as wheat bran for solid-state fermentation and soy flour and yeast extract for submerged fermentation. Subsequently, the enzyme was purified using ion exchange chromatography, and its biochemical, kinetic, and thermodynamic characterization was carried out. In the solid-state fermentation process, it was observed that production occurred successfully using 3 g of wheat bran with 20% moisture, resulting in an enzymatic activity of 41.36 U/mL. The protease demonstrated stability over a wide range of pH (from 5.0 to 10.0) and temperature (from 20°C to 30°C), with greatest activity at pH 7.0 and 50°C. In assays with specific ions and substrates, enzymatic activity was reduced in the presence of Cu2+ and inhibited by phenylmethylsulfonyl fluoride (PMSF), suggesting its behavior as a serine protease. The maximum rate of proteolytic activity was 140.0 U/mL, and the Michaelis-Menten constant was 11.6 mg/mL. The thermodynamic parameters revealed Gibbs free energy of 69.79 kJ/mol, enthalpy of 5.86 kJ/mol, and entropy of -214.39 J/mol.K, in addition to activation energy and standard enthalpy of 8.34 kJ /mol and 21.0 kJ/mol, respectively. However, thermodynamic parameters of thermo-inactivation indicated a reversible denaturation mechanism. Purifying the enzyme by chromatography resulted in a purification factor 7.2, corroborated by the presence of a single protein band of 14.7 kDa on SDS-PAGE. Furthermore, the optimized production of protease and collagenase was carried out by submerged fermentation, using an experimental design varying the soy flour and yeast extract concentration. The most efficient condition for producing these enzymes, with a soy flour concentration of 2.0% and yeast extract of 0.1%, was scaled from 50 mL (Erlenmeyer flask) to 1.5 L in a stirred tank bioreactor (STR). Fermentations conducted under these conditions resulted in exponential biomass growth between 20 and 42 hours, reaching a maximum growth rate (µmax) of 0.09 h^-1. Although the enzymes showed greater activity at 65 hours, collagenase production increased from 33.5 to 148.5 U/mL (+343%), while protease production decreased from 16.3 to 11.7 U/mL (-30%). The biomass conversion factors for protease and collagenase (Yx/p) were 2.70 and 34.22 U/mgx, respectively. These results demonstrate the potential of Aspergillus heteromorphus URM 0269 to produce protease and collagenase through two different fermentative processes and substrates, showing the possibility of using this fungus for enzyme production for future applications in industry.

COMMITTEE MEMBERS:
Presidente - TATIANA SOUZA PORTO
Interno - EMMANUEL VIANA PONTUAL
Externa à Instituição - MARIA DAS GRAÇAS CARNEIRO DA CUNHA - UFPE