Deletion of the Loop Linking Two Domains of Exo-Inulinase InuAMN8 Diminished the Enzymatic Thermo-Halo-Alcohol Tolerance

Inulin is the rich water-soluble storage polysaccharide after starch in nature, and utilization of inulin through hydrolysis of exo-inulinases has attracted much attention. Thermo-halo-alcohol tolerance is essential for exo-inulinase applications, while no report reveals the molecular basis involved in halo-alcohol tolerance of exo-inulinases via experimental data. In this study, two loops of exo-inulinase InuAMN8, including the loop built with (360)GHVRLGPQP(368) linking domains of Glyco_hydro_32N and Glyco_hydro_32C and another loop built with (169)GGAG(172) in the catalytic domain, were deleted to generate mutants MutG360 Delta 9 and MutG169 Delta 4, respectively. After heterologous expression, purification, and dialysis, InuAMN8, MutG169 Delta 4, and MutG360 Delta 9 showed half-lives of 144, 151, and 7 min at 50 degrees C, respectively. InuAMN8 and MutG169 Delta 4 were very stable, while MutG360 Delta 9 showed a half-life of approximately 60 min in 5.0% (w/v) NaCl, and they showed half-lives of approximately 60 min in 25.0, 25.0, and 5.0% (w/v) ethanol, respectively. Structural analysis indicated that two cation-pi bonds, which contributed to thermal properties of InuAMN8 at high temperatures, broke in MutG360 Delta 9. Four basic amino acid residues were exposed to the structural surface of MutG360 Delta 9 and formed positive and neutral electrostatic potential that caused detrimental effects on halo-alcohol tolerance. The study may provide a better understanding of the loop-function relationships that are involved in thermo-halo-alcohol adaptation of enzymes in extreme environment.