Relevance of aromatic and polar amino acids in the specificity of Inulinase ISO3 from Kluyveromyces marxianus: A molecular dynamics approach with an experimental verification.

The Inulinase from Kluyveromyces marxianus ISO3 (Inu-ISO3) is an enzyme able to hydrolyze linear fructans such as chicory inulin as well as branched fructans like agavin. This enzyme was cloned and expressed in Komagataella pastoris to study the role of selected aromatic and polar residues in the catalytic pocket by Alanine scanning. Molecular dynamics (MD) simulations and enzyme kinetics analysis were performed to study the functional consequences of these amino acid substitutions. Site-directed mutagenesis was used to construct the mutants of the enzyme after carrying out the MD simulations between Inu-ISO3 and its substrates. Mutation Trp79:Ala resulted in the total loss of activity when fructans were used as substrates, while with sucrose, the activity decreased by 98 %. In contrast, the mutations Phe113:Ala and Gln236:Ala increased the invertase activity when sucrose was used as a substrate. Although these amino acids are not part of the conserved motifs where the catalytic triad is located, they are essential for the enzyme's activity. In silico and experimental approaches corroborate the relevance of these residues for substrate binding and their influence on enzymatic activity.

Purification and characterization of an inulinase produced by a Kluyveromyces marxianus strain isolated from blue agave bagasse.

Exo-inulinases are versatile enzymes that have gained attention in recent years due to their ability to hydrolyze linear and branched polyfructose chains found in inulines. Agavin, a branched inulin, is found in Agave plant, the raw matter to produce tequila. Our group has isolated several microbial strains from agave bagasse, an agro-industrial residue from tequila production that increases yearly. Strain ISO3, identified as Kluyveromyces marxianus, showed a remarkable activity towards agavin, and from its fermentation liquor an inulinolytic enzyme (Inu-ISO3) was purified. The isolated enzyme is a glycosylated dimeric protein with a molecular mass of ~256 kDa, as determined by DLS and SEC. The enzyme has an isoelectric pH of 4.6 and has both inulinase and invertase activities with an I/S ratio (ratio of activity with agavin to activity with sucrose) of 1.39. The enzyme has temperature and pH optima of 50 °C and 5.5, respectively, and follows hyperbolic kinetics with agavin (kcat of 339 ± 27 s-1 and KM of 11.8 ± 1.5 mM). The remarkable activity of Inu-ISO3 on linear and branched inulin spotlights this enzyme as a potential player in the treatment of agricultural residua for the generation of added-value products.