Stabilization of elements of secondary structure in Aspergillus awamori exo-inulinase for thermostability improvement

Abstract

Enzymes with high thermostability provide bioconversion under elevated temperatures during industrial processes. Additional improvement in the enzyme thermostability allows further increasing the temperature for cost reduction. Aspergillus awamori exo-inulinase is sufficiently thermostable to hydrolyze inulin at temperatures up to 70 °C during the industrial process of bioconversion of the polysaccharide inulin into high-fructose syrup. Amino acid substitutions S322P and T521P were designed to improve the thermostability of A. awamori exo-inulinase and increase the temperature of the hydrolysis. The substitutions provided stabilization of α-helix and β-sheet as detected with molecular dynamics simulations at 70 °C. The substitution S322P also provided stabilization of the cleft of the active center, which correlated with the improvement in the thermostability of the enzyme. The variant S322P demonstrated a 1.5-fold increase in thermostability at 70 °C and a 2 °C increase in the temperature of inulin hydrolysis.