Characterization of a salt-tolerated exo-fructanase from Microbacterium sp. XL1 and its application for high fructose syrup preparation from inulin.

Exo-fructanase enzymes catalyze the hydrolysis of β-2,6 and β-2,1 linkages in levan and inulin fructans, respectively, yielding fructose. In this study, we identified a multidomain exo-fructanase, Mle3A, from Microbacterium sp. XL1. Mle3A is a 124.2 kDa protein comprising a GH32 N-terminal five-bladed β-propeller structure, a GH32 C-terminal β-sandwich module, and a fibronectin type 3 domain. The recombinant enzyme rMle3A exhibited peak activity at temperatures of 50-55 °C and a pH of 5.5, demonstrating hydrolytic capabilities towards levan, inulin, sucrose, and raffinose. The activity of rMle3A on inulin was enhanced in the presence of Mn, Ca, Ba, Sr, Co, and Mg ions. Notably, 5 mM Mn increased the inulin hydrolytic activity of rMle3A by over 187 %, and the enzyme's activity was unaffected by NaCl concentrations ranging from 0 to 3 M. Purified rMle3A was effectively utilized to produce high fructose syrup from inulin, achieving a maximum fructose concentration of 26.98 g/L and 71.9 % inulin hydrolysis under optimal conditions (85 rpm, 50 °C, pH 5.5) within 2.5 h. This study introduces a new salt-tolerant, multi-ion facilitated fructanase, rMle3A, for the conversion of inulin biomass into high fructose syrup and other high-value chemicals.