Cellulases are classically divided into three groups of enzymes, synergistically involved
in cellulose hydrolysis: endoglucanases (EC 3.2.1.4), exoglucanases (EC 3.2.1.91) and β-
glucosidases (EC 3.2.1.21). Cellulases belonging to all three groups are crucial for the
conversion of lignocellulosic materials and are useful in various industries. However,
complex cellulose structure and limitations of existing enzymes in cellulose degradation
restrict broader cellulase use in industrial applications. More efficient use of cellulases
for this purpose can be achieved with the improvement of their catalytic activity and
stability, as well as by understanding process-specific enzyme requirements and
engineering tailored enzymes for these processes. One way of improving cellulase
applicability is through oligomerization. Oligomeric enzymes often exhibit greater
resistance to extreme conditions; additionally, oligomerization can increase enzyme
adsorption to insoluble substrates. As part of this master’s thesis, we aimed to prepare a
tetrameric version of cellulase bglC, an endoglucanase from the bacterium Bacillus
subtilis, by using a thermostable p53 tetramerization domain (p53TD_Q331R) as a C-terminal fusion. We investigated whether the fusion protein oligomerises and whether the
change in oligomeric state influences its activity against the polymeric substrate
carboxymethylcellulose. We prepared the fusion protein construct by Gibson assembly
and purified both variants of the endoglucanase. We determined the oligomeric state with
size exclusion chromatography and mass photometry. Enzymatic activity of both variants
was subsequently analysed using the DNS assay, enabling the determination of the
corresponding kinetic parameters. We were unable to obtain the intended tetrameric form
of the full-length fusion protein, likely due to proteolytic cleavage of the fusion protein
in the interdomain region of cellulase bglC. Proteolytic cleavage and the absence of fusion
protein tetramerization could explain why the fusion protein exhibited lower apparent
activity compared to the recombinant wild-type variant of cellulase bglC.
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