| Summary: | <span style="font-variant: small-caps;">d</span>-Tagatose, a functional sweetener, is converted from <span style="font-variant: small-caps;">d</span>-galactose by <span style="font-variant: small-caps;">l</span>-arabinose isomerase, which catalyzes the conversion of <span style="font-variant: small-caps;">l</span>-arabinose to <span style="font-variant: small-caps;">l</span>-ribulose. In this study, the <i>araA</i> gene encoding <span style="font-variant: small-caps;">l</span>-arabinose isomerase from <i>Klebsiella pneumoniae</i> was cloned and expressed in <i>Escherichia coli</i>, and the expressed enzyme was purified and characterized. The purified <span style="font-variant: small-caps;">l</span>-arabinose isomerase, a soluble protein with 11.6-fold purification and a 22% final yield, displayed a specific activity of 1.8 U/mg for <span style="font-variant: small-caps;">d</span>-galactose and existed as a homohexamer of 336 kDa. The enzyme exhibited maximum activity at pH 8.0 and 40 °C in the presence of Mn<sup>2+</sup> and relative activity for pentoses and hexoses in the order <span style="font-variant: small-caps;">l</span>-arabinose > <span style="font-variant: small-caps;">d</span>-galactose > <span style="font-variant: small-caps;">l</span>-ribulose > <span style="font-variant: small-caps;">d</span>-xylulose > <span style="font-variant: small-caps;">d</span>-xylose > <span style="font-variant: small-caps;">d</span>-tagatose > <span style="font-variant: small-caps;">d</span>-glucose. The thermal stability of recombinant <i>E. coli</i> cells expressing <span style="font-variant: small-caps;">l</span>-arabinose isomerase from <i>K. pneumoniae</i> was higher than that of the enzyme. Thus, the reaction conditions of the recombinant cells were optimized to pH 8.0, 50 °C, and 4 g/L cell concentration using 100 g/L <span style="font-variant: small-caps;">d</span>-galactose with 1 mM Mn<sup>2+</sup>. Under these conditions, 33.5 g/L <span style="font-variant: small-caps;">d</span>-tagatose was produced from <span style="font-variant: small-caps;">d</span>-galactose with 33.5% molar yield and 67 g/L/h productivity. Our findings will help produce <span style="font-variant: small-caps;">d</span>-tagatose using whole-cell reactions, extending its industrial application.
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