Unit:
Specialty BiotechnologyLibrary of the School of Science
Author:
Maragkozoglou Elpida
Supervisors info:
Διομή Μαμμά Επίκουρη Καθηγήτρια Σχολή Χημικών Μηχανικών Εθνικό Μετσόβιο Πολυτεχνείο
Original Title:
Study of the synergistic action of cellulolytic enzymes of a genetic locus of the bacterium Bacillus safensis
Translated title:
Study of the synergistic action of cellulolytic enzymes of a genetic locus of the bacterium Bacillus safensis
Summary:
In this study, a β-1,4-exoglucanase of the GH48 protein family was characterized and its synergistic action with an endo-1,4-β-D-glucanase of the GH9 protein family and a β-glucanase of the GH5 protein family was examined. These three enzymes originate from a Bacillus safensis (ATHUBA63), which was isolated from a local soil sample. The bsgh48 gene was cloned and overexpressed in competent cells of Escherichia coli BL21. From these bacterial cells, BsGH48 was isolated. The molecular weight of BsGH48 was estimated at 78.6 kDa. A bioinformatics analysis took place where the bsgh48 sequence was compared with others from online databases and the protein's 3D–structure was predicted. The optimum conditions of action for BsGH48 were determined at 50 °C and pH 5.0. The effect of several modulators on the activity of BsGH8 was examined. The Mn2+ and Ca2+ divalent ions enhance substantially its activity. However, it is significantly reduced by both ferrous ions, Fe2+ and Fe3+. BsGH48 was not affected by SDS or EDTA. The stability of the protein molecule was tested. From the results of the thermal stability assay the thermal inactivation energy (E(a)d) of BsGH48 was calculated at 159.7 ± 7.3 kJ/mole. Its thermodynamic parameters of thermal deactivation were also evaluated. At the pH stability assay, BsGh48 showed great stability in the pH range of 5.0-9.0. The substrates that BsGH48 hydrolyzed are Avicel and PASC. The kinetic constants, Km and Vmax, indicated that BsGh48 presents a preference towards PASC as a substrate. Moreover, BsGH48 proved to be a non-processive enzyme. The hydrolysis products of PASC from BsGH48, BsGH9, and BsGH5 were analyzed with HPAEC-PAD, to set a backbone for the synergistic study. BsGH48’s only product was cellobiose. BsGH9’s products were cellobiose and glucose. BsGH5’s products were cellobiose, cellotriose, and cellotetraose. BsGH9 presented the highest enzymatic activity. In the synergistic study, the enzymes were first paired up and then they were all included in the reaction mix. From their synergistic action, there were not produced any new products. Nevertheless, the enzymatic activity of all the enzymes and the hydrolysis of PASC were
enhanced.
Main subject category:
Science
Keywords:
cellulases, Bacillus safensis, β-1,4-exoglucanase, endo-1,4-β-D-glucanase, β-glucanase, synergism
File:
File access is restricted until 2024-08-16.
Master_thesis_E.M.pdf
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File access is restricted until 2024-08-16.
