Cloning, expression and characterization of xylose isomerase from Euphausia superba

A novel xylose isomerase gene, with an open reading frame (ORF) of 1368 bp, was cloned from Euphausia superba and successfully expressed in Escherichia coli (E. coli). The enzyme catalyzes the isomerization of D-xylose into a rare sugar D-xylulose. The purified protein (EsXLI), with a molecular weight of approximately 51 kDa, was obtained via Ni²⁺-NTA affinity chromatography. The catalytic efficiency (k_cat/K_m) and K_m for D-xylose were 0.3011 mM^-1·s^-1 and 67.99 mM, respectively, while for d-glucose, were 0.0286 mM^-1·s^-1 and 216.38 mM, respectively. Compared to other xylose isomerases with optimum temperatures above 80 °C, EsXLI's optimum reaction temperature was 50 °C, with an optimum pH of 7.0. Co²⁺, Mn²⁺, and Mg²⁺ ions enhanced its catalytic activity, whereas Ba²⁺, Ca²⁺, ethylenediaminetetraacetic acid (EDTA), and β-Mercaptoethanol (β-ME) inhibited it. The EsXLI-K253R mutant showed a 1.25-fold increase in activity compared to the wild-type, while the EsXLI-D328A mutant demonstrated improved low-temperature performance, with its optimum temperature decreased from 50 to 40 °C. These findings suggest that xylose isomerase from E. superba is a promising candidate for the production of rare sugars due to its industrially competitive temperature.