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 360GHVRLGPQP368 linking domains of Glyco_hydro_32N and Glyco_hydro_32C and another loop built with 169GGAG172 in the catalytic domain, were deleted to generate mutants MutG360Δ9 and MutG169Δ4, respectively. After heterologous expression, purification, and dialysis, InuAMN8, MutG169Δ4, and MutG360Δ9 showed half-lives of 144, 151, and 7 min at 50°C, respectively. InuAMN8 and MutG169Δ4 were very stable, while MutG360Δ9 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-π bonds, which contributed to thermal properties of InuAMN8 at high temperatures, broke in MutG360Δ9. Four basic amino acid residues were exposed to the structural surface of MutG360Δ9 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.

Enzymatic preparation of manno-oligosaccharides from locust bean gum and palm kernel cake, and investigations into its prebiotic activity

BACKGROUND: Manno-oligosaccharides (MOS) is known as a kind of prebiotics. Mannanase plays a key role for the degradation of mannan to produce MOS. In this study, the mannanases of glycoside hydrolase (GH) families 5 Man5HJ14 and GH26 ManAJB13 were employed to prepare MOS from locust bean gum (LBG) and palm kernel cake (PKC). The prebiotic activity and utilization of MOS were assessed in vitro using the probiotic Lactobacillus plantarum strain. RESULTS: Galactomannan from LBG was converted to MOS ranging in size from mannose up to mannoheptose by Man5HJ14 and ManAJB13. Mannoheptose was got from the hydrolysates produced by Man5HJ14, which mannohexaose was obtained from LBG hydrolyzed by ManAJB13. However, the same components of MOS ranging in size from mannose up to mannotetrose were observed between PKC hydrolyzed by the mannanases mentioned above. MOS stability was not affected by high-temperature and high-pressure condition at their natural pH. Based on in vitro growth study, all MOS from LBG and PKC was effective in promoting the growth of L. plantarum CICC 24202, with the strain preferring to use mannose to mannotriose, rather than above mannotetrose. CONCLUSIONS: The effect of mannanases and mannan difference on MOS composition was studied. All of MOS hydrolysates showed the stability in adversity condition and prebiotic activity of L. plantarum, which would have potential application in the biotechnological applications.