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

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

Alignment of GalOx from <i>F. oxysporum</i> and <i>F. graminearum</i>.

<p>The prepro sequence is underlined. Amino acid residues involved in copper binding are highlighted.</p

3D Structure of GalOx of <i>F. oxysporum</i>.

<p>A: Overall structure showing the predominantly β-structure. The N-terminus, C-terminus and the copper atom in the active site are highlighted. B: The active site of GalOx showing the copper ligands and the thioether cross-link. The structural model was generated by homology modelling based on the published structure of mature GalOx from <i>F. graminearum</i> (PDB 1gog) using SWISS_MODEL.</p

Effect of the pH on the activity of GalOx expressed in <i>E. coli</i>.

<p>The buffers used were 50(♦), 50 mM phosphate (_▀) and 50 mM Tris (▴).</p

Temperature dependence of the activity of GalOx expressed in <i>E. coli.</i>

<p>Temperature dependence of the activity of GalOx expressed in <i>E. coli.</i></p

Purification of recombinant GalOx expressed in <i>E. coli.</i>

<p>Purification of recombinant GalOx expressed in <i>E. coli.</i></p

MALDI-TOF peptide mass map of GalOx expressed in <i>E. coli</i>.

<p>The peptides were generated by sequential digestion using trypsin and Asp-N. The peak labels correspond to the [M+H]⁺ ions of the obtained peptide fragments and their positions in the GalOx sequence. The spectrum also shows two intense signals (marked with asterisk) at m/z 2237.9 and 2374.9 related to the cross-linked peptides 266–274/312–323 and 265–274/312–323, respectively. The identity of the cross-linked peptides was verified by MS/MS fragmentation.</p

MALDI-FTMS data of the cross-linked peptides produced by in-gel digestion of GalOx. The cross-linked amino acids C230 and Y274 are highlighted.

<p>MALDI-FTMS data of the cross-linked peptides produced by in-gel digestion of GalOx. The cross-linked amino acids C230 and Y274 are highlighted.</p

Apparent kinetic constants of GalOx produced in <i>E. coli</i> for several electron donor substrates.

<p>Apparent kinetic constants of GalOx produced in <i>E. coli</i> for several electron donor substrates.</p

Electrophoresis analysis of crude extract and purified GalOx expressed in <i>E. coli</i>.

<p>Lane 1, Precision Plus Protein Standard (BioRad); lane 2, GalOx crude extract; lane 3, GalOx after IMAC; lane 4, GalOx after size-exclusion chromatography.</p