l‑Arabinose Isomerase and d‑Xylose Isomerase from Lactobacillus reuteri: Characterization, Coexpression in the Food Grade Host Lactobacillus plantarum, and Application in the Conversion
of d‑Galactose and d‑Glucose
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Abstract
The l-arabinose isomerase (l-AI) and the d-xylose
isomerase (d-XI) encoding genes from Lactobacillus
reuteri (DSMZ 17509) were cloned and
overexpressed in Escherichia coli BL21
(DE3). The proteins were purified to homogeneity by one-step affinity
chromatography and characterized biochemically. l-AI displayed
maximum activity at 65 °C and pH 6.0, whereas d-XI showed
maximum activity at 65 °C and pH 5.0. Both enzymes require divalent
metal ions. The genes were also ligated into the inducible lactobacillal
expression vectors pSIP409 and pSIP609, the latter containing a food
grade auxotrophy marker instead of an antibiotic resistance marker,
and the l-AI- and d-XI-encoding sequences/genes
were coexpressed in the food grade host Lactobacillus
plantarum. The recombinant enzymes were tested for
applications in carbohydrate conversion reactions of industrial relevance.
The purified l-AI converted d-galactose to d-tagatose with a maximum conversion rate of 35%, and the d-XI isomerized d-glucose to d-fructose with a maximum
conversion rate of 48% at 60 °C