Unraveling the potential of uninvestigated thermoalkaliphilic lipases by molecular docking and molecular dynamic simulation: an in silico characterization study

Thermoalkaliphilic lipase enzymes are mostly favored for use in the detergent industry. While there has been considerable research on Geobacillus lipases, a significant portion of these enzymes remains unexplored or undocumented in the scientific literature. This work performed in silico phylogeny, sequence alignment, structural and enzyme–substrate interaction analyses of the five thermoalkaliphilic lipases belonging to different Geobacillus species ( Geobacillus stearothermophilus lipase = Gs Lip, Geobacillus sp. B4113_201601 lipase = Gb4 Lip, Geobacillus kaustophilus HTA426 lipase = Gk Lip, Geobacillus sp. SP22 lipase = Gsp Lip, Geobacillus sp. NTU 03 lipase = Gnt Lip). For this purpose, unreviewed enzyme sequences of five Geobacillus thermoalkaliphilic lipases were analyzed at sequence and phylogeny levels. 3D homology enzyme models were built, validated, and investigated by different bioinformatics tools. The ligand interactions screening using seven para-nitrophenyl ( p NP) esters and enzyme–ligand interactions were analyzed on Gb4 Lip: p NP-C12 and BTL2: p NP-C12 by MD simulation. Biophysicochemical characteristic analysis showed that Gb4 Lip had a theoretical T m value of above 65 ºC, and a higher aliphatic index indicating greater thermal stability. Sequence alignment showed a hydrophilic threonine in the α6 helix of Gb4 Lip, indicating high enzymatic activity. A normalized temperature factor B (B’-factor) analysis showed that the lid domains of five lipases significantly possessed lower B’-factor values, compared to G. thermocatenulatus lipase 2 (BTL2), indicating that they had higher rigidity. Molecular docking results indicated that the five lipases had the highest binding affinity toward p NP-C12. The RMSF investigation revealed that the thermostability of Gb4 Lip is influenced by specific molecular elements: D202-S203 within the αB region of the lid domain, and E274-Q275 within the b3 strand, as well as W278 in the b3–b4 loop, and H282 in the b4 strand of the Ca 2+ -binding region. MD simulation analysis showed that catalytic residue S114 and at least one oxyanion hole residue (F17 and/or Q114) in Gb4 Lip frequently formed hydrogen bonds with the p NP-C12 ligand at 343 K and 348 K throughout the simulation process, indicating that Gb4 Lip might catalyze relatively long-chain ligand p NP-C12 with high performance. In conclusion, Gb4 Lip might be a more suitable candidate as the detergent additive. In addition, this investigation can offer