Biochemical and structural characterization of a family-9 glycoside hydrolase bioprospected from the termite Syntermes wheeleri gut bacteria metagenome

Abstract

Glycosyl hydrolases (GH) are enzymes involved in the degradation of plant biomass. They are important for biorefineries that aim at the sustainable utilization of lignocellulosic residues to generate value-added products. The termite Syntermes wheeleri gut microbiota showed an abundance of bacteria from the phylum Firmicutes, a phylum with enzymes capable of breaking down cellulose and degrading lignin, facilitating the use of plant materials as a food source for termites. Using bioinformatics techniques, cellobiohydrolases were searched for in the gut metagenome of the termite Syntermes wheeleri, endemic to the Cerrado. After selecting sequences of the target enzymes, termite gut microbiome metatranscriptome data were used as the criteria to choose the GH9 enzyme sequence Exo8574. Here we present the biochemical and structural characterization of Exo8574, a GH9 enzyme that showed activity with the substrate p-nitrophenyl-D-cellobioside (pNPC), consistent with cellobiohydrolase activity. Bioinformatics tools were used to perform phylogeny studies of Exo8574 and to identify conserved families and domains. Exo8574 showed 48.8 % homology to a protein from a bacterium belonging to the phylum Firmicutes. The high-quality three-dimensional (3D) model of Exo8574 was obtained by protein structure prediction AlphaFold 2, a neural network-based method. After the heterologous expression of Exo8574 and its purification, biochemical experiments showed that the optimal activity of the enzyme was at a temperature of 55 ºC and pH 6.0, which was enhanced in the presence of metal ions, especially Fe^2 +. The estimated kinetic parameters of Exo8574 using the synthetic substrate p-nithrophenyl-beta-D-cellobioside (pNPC) were: V_max = 9.14 ± 0.2 x10⁻⁵ μmol/min and K_m = 248.27 ± 26.35 μmol/L. The thermostability test showed a 50 % loss of activity after 1 h incubation at 55 °C. The secondary structure contents of Exo8574 evaluated by Circular Dichroism were pH dependent, with greater structuring of protein in β-antiparallel and α-helices at pH 6.0. The similarity between the CD results and the Ramachandran plot of the 3D model suggests that a reliable model has been obtained. Altogether, the results of the biochemical and structural characterization showed that Exo8574 is capable of acting on p-nithrophenyl-beta-D-cellobioside (pNPC), a substrate that mimics bonds cleaved by cellobiohydrolases. These findings have significant implications for advancing in the field of biomass conversion while also contributing to efforts aimed at overcoming challenges in developing more efficient cellulase cocktails.