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

Mannan biotechnology: from biofuels to health

<div><p></p><p>Mannans of different structure and composition are renewable bioresources that can be widely found as components of lignocellulosic biomass in softwood and agricultural wastes, as non-starch reserve polysaccharides in endosperms and vacuoles of a wide variety of plants, as well as a major component of yeast cell walls. Enzymatic hydrolysis of mannans using mannanases is essential in the pre-treatment step during the production of second-generation biofuels and for the production of potentially health-promoting manno-oligosaccharides (MOS). In addition, mannan-degrading enzymes can be employed in various biotechnological applications, such as cleansing and food industries. In this review, fundamental knowledge of mannan structures, sources and functions will be summarized. An update on various aspects of mannan-degrading enzymes as well as the current status of their production, and a critical analysis of the potential application of MOS in food and feed industries will be given. Finally, emerging areas of research on mannan biotechnology will be highlighted.</p></div

Data collection and crystallographic refinement statistics for mutant P2O structures with fluorinated galactoses.

1<p>The outer shell statistics of the reflections are given in parentheses. Shells were selected as defined in XDS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086736#pone.0086736-Kabsch1" target="_blank">[36]</a> by the user.</p>2<p>R_sym = [Σ_hkl Σ_i |I–<i>|/Σ_hkl Σ_i |I| ]×100%.</i></p><i>3<p>CC(1/2) = Percentage of correlation between intensities from random half-datasets. Values given represent correlations significant at the 0.1% level <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086736#pone.0086736-Karplus1" target="_blank">[44]</a>.</p>4<p>R_factor = Σ_hkl | |F_o|–|F_c| |/Σ_hkl |F_o|.</p>5<p>We note that the R and R_free values are suspiciously high a 1.9-Å resolution model with electron density of good quality. Data sanity tests excluded twinning, pseudotranslation, and misindexing as possible reasons. However, the data suffer from poor completeness in the low-resolution region and contain several ice rings, which may account for the problems encountered during refinement.</p>6<p>As determined by MolProbity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086736#pone.0086736-Lovell1" target="_blank">[45]</a>.</p></i

Structural Basis for Binding of Fluorinated Glucose and Galactose to <i>Trametes multicolor</i> Pyranose 2-Oxidase Variants with Improved Galactose Conversion

<div><p>Each year, about six million tons of lactose are generated from liquid whey as industrial byproduct, and optimally this large carbohydrate waste should be used for the production of value-added products. <i>Trametes multicolor</i> pyranose 2-oxidase (<i>Tm</i>P2O) catalyzes the oxidation of various monosaccharides to the corresponding 2-keto sugars. Thus, a potential use of <i>Tm</i>P2O is to convert the products from lactose hydrolysis, D-glucose and D-galactose, to more valuable products such as tagatose. Oxidation of glucose is however strongly favored over galactose, and oxidation of both substrates at more equal rates is desirable. Characterization of <i>Tm</i>P2O variants (H450G, V546C, H450G/V546C) with improved D-galactose conversion has been given earlier, of which H450G displayed the best relative conversion between the substrates. To rationalize the changes in conversion rates, we have analyzed high-resolution crystal structures of the aforementioned mutants with bound 2- and 3-fluorinated glucose and galactose. Binding of glucose and galactose in the productive 2-oxidation binding mode is nearly identical in all mutants, suggesting that this binding mode is essentially unaffected by the mutations. For the competing glucose binding mode, enzyme variants carrying the H450G replacement stabilize glucose as the <i>α</i>-anomer in position for 3-oxidation. The backbone relaxation at position 450 allows the substrate-binding loop to fold tightly around the ligand. V546C however stabilize glucose as the <i>β</i>-anomer using an open loop conformation. Improved binding of galactose is enabled by subtle relaxation effects at key active-site backbone positions. The competing binding mode for galactose 2-oxidation by V546C stabilizes the <i>β</i>-anomer for oxidation at C1, whereas H450G variants stabilize the 3-oxidation binding mode of the galactose <i>α</i>-anomer. The present study provides a detailed description of binding modes that rationalize changes in the relative conversion rates of D-glucose and D-galactose and can be used to refine future enzyme designs for more efficient use of lactose-hydrolysis byproducts.</p></div

Details of oxidation-binding modes for 2- or 3-fluorinated glucose.

a<p>The following three criteria are considered consistent with a productive binding mode: (i) The sugar is oriented for oxidation at C2; (ii) the substrate-binding loop is in the semi-open conformation; (iii) the side chain Oγ1 group of Thr169 is pointing away from the flavin N(5)/O(4) locus.</p>b<p>PDB code 3PL8 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086736#pone.0086736-Tan1" target="_blank">[19]</a>.</p>c<p>PDB code 2IGO <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086736#pone.0086736-Kujawa1" target="_blank">[14]</a>.</p>d<p>The ability of P2O variants to stabilize either the α- or β-anomer is uncorrelated to space group and crystal contacts, and depends solely on the new structural context provided by the mutation.</p>e<p>Italicized interactions represent interactions with the catalytic residues.</p

Principal productive and competing binding modes for fluorinated glucose and galactose.

<p>Structural overlay of the active sites in <i>Tm</i>P2O V546C (beige) and H450G (green). The V546C mutant is used as a reference since it displays the same binding modes as the H167A mutant, and for 2- or 3-fluorinated glucose, also agrees with the binding modes observed for the wild type. (a) Binding of <i>3F</i>Glc in the productive 2-oxidation binding mode. The sugar is stabilized as the <i>β</i>-anomer with O2 coordinated by His458 and Asn593 and C2 appropriately positioned for oxidation. The substrate-binding loop is in the semi-open conformation positioning Phe454 closely packed against the pyranose as has been described for the productive binding mode earlier <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086736#pone.0086736-Tan1" target="_blank">[19]</a>. (b) <i>2F</i>Glc in the competing 3-oxidation binding mode. In H450G, the C1 hydroxyl in <i>2F</i>Glc is stabilized in axial configuration (<i>α</i>-anomer) by Asp452 and Thr169 and the substrate-binding loop assumes the semi-open conformation. V546C stabilizes the <i>β</i>-anomer and reveals the open conformation of the substrate-binding loop as observed earlier for H167A in complex with <i>2F</i>Glc <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086736#pone.0086736-Kujawa1" target="_blank">[14]</a>. (c) <i>3F</i>Gal in productive 2-oxidation binding mode with the axial C4 hydroxyl group coordinated by Asp452 and Thr169. The substrate-binding loop is in the semi-open conformation. (d) <i>2F</i>Gal in competing binding modes. The competing binding mode observed for V546C corresponds to the <i>2F</i>Gal <i>β</i>-anomer oriented for oxidation at C1. The competing binding mode for H450G shows the <i>α-</i>anomer of <i>2F</i>Gal oriented for oxidation at C3. In both cases, the substrate-binding loop assumes the semi-open conformation compatible with the productive sugar-oxidation mode. All structures that bind sugar substrate show the Thr169 Oγ1 atom pointing <i>away</i> from the flavin N(5)/O(4) locus, which constitutes an additional hallmark of the productive binding mode. For clarity, the covalent link between the flavin and His167 is not shown in the pictures. The pictures were produced using the program PyMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086736#pone.0086736-DeLano1" target="_blank">[43]</a>.</p

Apparent steady-state kinetic constants of TmP2O wild-type and mutants with D-glucose or D-galactose as electron donor, and O₂ (air) under saturation as electron acceptor.

a<p>Since H167A was not designed to specifically improve galactose turnover, but to slow down flavin reduction during the reductive half-reaction, the kinetic constants for H167A were not determined. The mutant was included since it is a close structural mimic of the wild-type when sugar binding is concerned. Its purpose is therefore mainly for structural comparisons.</p

Comparison of conformation of the substrate-binding loop in <i>Tm</i>P2O complexes with fluorinated sugars.

<p>Superposition of mutant structures emphasizing the conformation of the substrate-binding loop. The FAD molecule and the pyranose sugar are shown as ball-and-stick models. (a) <i>Tm</i>P2O variants complexed with <i>3F</i>Glc corresponding to the productive 2-oxidation binding mode with the substrate-binding loop in the semi-open conformation. The relaxation induced by the H450G replacement (green and light-blue models) is highlighted by a shaded circle. The H167A model corresponds to PDB code 3PL8 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086736#pone.0086736-Tan1" target="_blank">[19]</a>. (b) Mutant complexes with bound <i>2F</i>Glc in the competing 3-oxidation binding mode. The H167A (PDB code 2IGO <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086736#pone.0086736-Kujawa1" target="_blank">[14]</a>) and V546C variants show the open loop conformation, which also has been observed for the wild type. H450G and H450G/V546C show the productive semi-open loop conformation. (c) Mutant complexes of V546C, H450G and H450G/V546C with bound <i>3F</i>Gal in the productive 2-oxidation binding mode with the substrate-binding loop in the semi-open conformation. The wild-type mimic H167A displays did not bind the sugar and displays the closed, occluded loop conformation that is typically observed for <i>Tm</i>P2O in the absence of oxidizable sugar. The closed loop conformation is incompatible with sugar binding. (d) Mutant complexes with bound <i>2F</i>Gal. Despite the fundamentally different competing modes observed for H167A and V546C (C1-oxdiation mode) and H450G and H450G/V546C (C3-oxidation mode), all complexes show the substrate-binding loop in the semi-open loop conformation associated with productive sugar binding. The pictures were produced using the program PyMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086736#pone.0086736-DeLano1" target="_blank">[43]</a>.</p

Oxidation of methionines.

<p>Methionine-containing peptide fragments detected that show a mass increase of 16 or 32 Da after proteolytic digestion of <i>Tm</i>POx (inactivated during turnover of 100 mM D-glucose, treated with endogenous H₂O₂ or unaffected). Proteolytic digestion was performed with trypsin or Asp-N protease as indicated. The last column indicates whether the particular Met is considerably oxidised during substrate turnover and by H₂O₂ treatment compared to the unaffected sample (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148108#pone.0148108.g002" target="_blank">Fig 2</a>).</p

Active-site geometry of pyranose oxidase from <i>T</i>. <i>multicolor</i>.

<p>In the closed form of the active-site loop of pyranose oxidase from <i>T</i>. <i>multicolor</i> (<i>Tm</i>POx, PDB code 1TT0; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148108#pone.0148108.ref003" target="_blank">3</a>]), which is thought to be relevant for the oxidative half-reaction of POx [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148108#pone.0148108.ref003" target="_blank">3</a>], Phe454 is positioned in the direct vicinity of the isoalloxazine ring and the C4a/N5 locus, at which oxygen is reduced. The figure was generated using PyMOL (<a href="http://www.pymol.org/" target="_blank">http://www.pymol.org/</a>).</p