In silico characterization of thermoacidophilic alpha-L-arabinofuranosidaze enzymes of bacterial origin

The aim of this study was to perform in silico characterization of bacterial alpha-L-arabinofuranosidase enzymes with four thermoacidophilic properties and to identify the bacterial thermoacidophilic alpha-L-arabinofuranosidase with the best properties based on these results. For this purpose, the amino acid sequences of unreviewed alpha-L-arabinofuranosidase enzymes in the UniProt/TrEMBL database were screened and four enzymes with the highest thermoacidophilic properties were selected based on theoretical pI, theoretical Tm, aliphatic index and instability index values. In silico characterization of these four enzymes was performed through sequence and structure level analyses. The analysis showed that arabinofuranosidase enzymes exhibited a Tm above 65°C. In addition, their isoelectric points (pI) decreased to the range of 4.57 to 4.72. In this study, the Geobacillus stearothermophilus Abf enzyme (Q9XBQ3) operating at an optimum temperature and pH of 70oC and 6.0, respectively, had a pI of 5.56 and a Tm range of 55-65°C, while the Acetivibrio thermophilus Abf enzyme (Q9XBQ3) operating optimally at pH 6.0 and 60°C (Taylor et al, 2006) from Acetivibrio thermocellus (A3DIH0), operating optimally at pH 6.0 and 60°C, appeared to have a pl of 5.43 and a Tm range of 55-65°C. In addition, the aliphatic indices of Abf enzymes (85.45-93.41) were comparable to AbfA and A3DIH0 and the instability indices were lower than AbfA and A3DIH0. ClAbf was found to have the highest aliphatic index among the four Abf. Phylogenetic study to find the evolutionary relationship of the four enzymes revealed that all four Abf enzymes were categorized under the GH51 family. The alphafold model was used for structure prediction. The predicted models were qualitatively characterized using various tools. The secondary structures of the Abf models obtained through stride were depicted, again the results from the Procheck plot showed that a significant percentage of residues in Abfs (ranging from 89.3% to 91.1%) were positioned in the most favorable regions. The overall quality of the alphafold models for arabinofuranosidases was assessed using ProSA's Z-scores. The results revealed Z-scores ranging from -9.64 to -10.62. Verify3D evaluation confirmed an average 3D-1D score ≥ 0.2 for 82.63-88.87% of amino acids in all Abf structures. Abfs exhibited Qualitative Model Energy Analysis of Energy (QMEAN) scores approaching zero, ranging from -0.34 to 0.19, indicating a high level of similarity to native structures. According to BANΔIT analysis, the B'-factor profiles of all four Abfs exhibited similarities. All four Abfs had lower B'-factors in 21 regions (3-23) compared to AbfA. Keywords: In silico, thermoacidophilic, animal feed additive, alpha-L-arabinofuranosidase, industrial enzyme