Banca de DEFESA: MONIZY DA COSTA SILVA

Uma banca de DEFESA de DOUTORADO foi cadastrada pelo programa.
STUDENT : MONIZY DA COSTA SILVA
DATE: 30/10/2023
TIME: 14:00
LOCAL: Google Meet
TITLE:

Production, purification, characterization and biotechnological applications of fungal hydrolases in agroindustrial residues.

KEY WORDS:

Hydrolase enzymes; Solid state fermentation; Purification; Biotechnological applications.

PAGES: 168
BIG AREA: Ciências Exatas e da Terra
AREA: Química
SUMMARY:

Brazilian agriculture, driven by technological advances and environmental concerns, is becoming an important engine of development. Research is being carried out to harness non-commercial raw materials such as lignocellulosic biomass to produce useful enzymes through solid-state fermentation (FES) with fungi such as Pleurotus djamor PLO13. Two of these enzymes, proteases and endoglucanases, have applications in diverse industries and are widely produced globally. Therefore, the present research aims to produce, purify, characterize and evaluate possible biotechnological applications of the protease and endoglucanase enzymes from the solid state fermentation process with the fungus P. djamor PLO13. Initially, a screening of four agro-industrial residues (BCA, FC, CA and FT) was carried out as support for the growth of the fungus, and FT proved to be the most efficient after 120 hours of cultivation. Subsequently, enzymatic assays were carried out to analyze the presence of protease and endoglucanase in the extract, and both were detected. The crude enzyme extract reached its maximum caseinolytic activity after 120 hours of cultivation. The specificity of the protease was analyzed in relation to the substrates tested and its inhibition in the presence of specific inhibitors, which allowed it to be classified as a serine protease similar to metal iondependent elastase-2-like, with a specific activity of 3,976 U.mg −1. This enzyme demonstrated high efficiency in the coagulation of pasteurized whole and reconstituted skimmed milk, both in the presence and absence of calcium, even at room temperature. The coagulation process was influenced by factors such as temperature, time and calcium concentration, which were thoroughly analyzed. As for the endoglucanase present in the crude extract, it was classified as halotolerant and halophilic and had an optimum temperature and pH of 50 °C and 5.0, respectively. Furthermore, it remained stable under conditions of 40 to 60 °C and pH 4.0 to 10.0, as well as in the presence of surfactant agents, such as SDS, triton X-100, Tween 40 and Tween 80. Its enzymatic activity it was activated in the presence of EDTA, Na+, ethanol, propanone, dichloromethane, dinitromethane, urea and β-mercaptoethanol, while Mg2+, Ca2+ and Cu2+, methanol and heptane reduced or inactivated it. For the purification of endoglucanase, an organic fractionation process followed by anion exchange chromatography was used. During this process, an enzyme with a molecular mass of approximately 26 kDa was purified and showed a remarkable recovery of 110%. Pure endoglucanase showed excellent performance in terms of temperature and pH, with optimum values of 50 °C and 5.0, respectively. Furthermore, it was stable over a wide range of temperatures, ranging from 30 to 70 °C, and pH, ranging from 3.0 to 10.0. This remarkable stability under varying conditions makes it especially valuable in industrial processes and it has been classified as halotolerant and halophilic. Furthermore, the enzymatic activity was activated by the presence of EDTA and Mn2+, while the presence of Mg2+ inhibited its activity. In terms of enzymatic kinetics, the enzyme exhibited a Km of 0.0997 ± 0.0063 mg/ml and a Vmax of 0.1122 ± 0.00109 mol/min/mL, indicating its effectiveness in the hydrolysis of specific substrates. These results indicate a high potential for industrial application for these enzymes, especially in the dairy, second-generation ethanol production and detergent manufacturing sectors, due to their stability under a wide range of conditions.

COMMITTEE MEMBERS:
Externo(a) à Instituição - ALDENIR FEITOSA DOS SANTOS - UNEAL
Interno(a) - 2089586 - FRANCIS SOARES GOMES
Presidente - 1811274 - HUGO JUAREZ VIEIRA PEREIRA
Interno(a) - 1294906 - RUTH RUFINO DO NASCIMENTO
Externo(a) ao Programa - 1488396 - TICIANO GOMES DO NASCIMENTO - null