Enzymatic Properties and Structural Insights Into the Derhamnosylating Alkaline α-l-Rhamnosidase From Aspergillus flavus.

Abstract

α-l-Rhamnosidases are ubiquitous enzymes responsible for derhamnosylation of α-l-rhamnose moiety from a variety of glycoconjugates and numerous natural glycosides. An α-l-rhamnosidase-secreting fungal strain was isolated from soil sample. Further, it was identified as Aspergillus flavus through internal transcribed spacer (ITS) gene sequencing. The enzyme was purified to homogeneity using ion-exchange and gel filtration chromatography and exhibited molecular weight of 71 ± 1 kDa. The maximum catalytic efficiency for the α-l-rhamnosidase was established to be pH 10.0 and at a temperature of 50°C. The purified enzyme exhibits a K_m 0.41 ± 0.06 mM and a V_max 2.43 ± 0.17 µmol/min/mg for naringin hydrolysis. In this study, we modeled the 3D structure of A. flavus α-l-rhamnosidase using SWISS Model and validated it via PDBsum and PROCHECK. Molecular docking of A. flavus α-l-rhamnosidase with naringin and p-nitrophenyl-α-l-rhamnopyranoside (pNPR) identified key binding interactions. Electrostatic surface analysis highlighted ligand-binding sites, revealing crucial residues for substrate recognition and enzyme stability. Active site residues of A. flavus α-l-rhamnosidase forming hydrogen bonds and hydrophobic interactions with naringin and pNPR were identified, providing insights into substrate specificity and its potential applications in glycoside hydrolysis.