Results of shake flask studies (36 h) for <i>de novo</i> conversion of 1,2-diols using resting cells of recombinant <i>E</i>. <i>coli</i> strains.

<p>Results of shake flask studies (36 h) for <i>de novo</i> conversion of 1,2-diols using resting cells of recombinant <i>E</i>. <i>coli</i> strains.</p

General reaction scheme for glycerol/1,2-diols conversion to corresponding alcohol and acid by lumen enzymes of <i>pdu</i>-microcompartment.

<p>General reaction scheme for glycerol/1,2-diols conversion to corresponding alcohol and acid by lumen enzymes of <i>pdu</i>-microcompartment.</p

Time course of biotransformation of glycerol and 1,2-PDO using wild-type and mutant <i>L</i>. <i>reuteri</i>.

<p>Time course of biotransformation of glycerol and 1,2-PDO using wild-type and mutant <i>L</i>. <i>reuteri</i>.</p

Redesign of glycerol dehydratase from <i>L</i>. <i>reuteri</i>.

<p>A) Overall fold of the glycerol dehydratase monomer and the proposed biological dimer; Stereodrawing of the B) 1,2-PD or C) glycerol bound form of the glycerol dehydratase. Homology modeling for the prediction of tertiary structure of glycerol dehydratase (PduCDE) resulted in a model based on the crystal structure of diol dehydratase-cyanocobalamin complex from <i>Klebsiella oxytoca</i> (1DIO), with sequence similarities and coverages above 42% and 95%, respectively. Quality model assessment of the homology model revealed a QMEAN6-score of 0.83 and a Z-score of -0.53. Ramachandran plot revealed that none of residues were present in the disallowed regions. Color codes for carbon atoms: yellow for adenosylcobalamin (AdoCbl), white for glycerol/1,2-PD, purple for calcium ion. The model figure was generated using Pymol. D) Sequence alignment of large subunit of glycerol dehydratase (PduC) from different bacteria. The numbering scheme follows the amino acid sequences of PduCs. Identical residues in all sequences are highlighted in black and conserved in grey. E) Relative activities of WT/mutant glycerol dehydratases towards different 1,2-diols/glycerol. The data is normalized by taking the activity of the wild-type enzyme towards glycerol as 100%.</p

Kinetic analysis of lumen enzymes towards different substrates.

<p>A) Relative activities of cell free extracts of recombinant <i>E</i>. <i>coli</i> expressing wild type glycerol dehydratase toward glycerol/1,2-diols, using the activity towards glycerol as 100%. B) <i>K</i>_m values for PduQ and PduP towards different aldehydes. Vmax values for C) PduQ and D) PduP.</p

Immobilization to Positively Charged Cellulose Nanocrystals Enhances the Antibacterial Activity and Stability of Hen Egg White and T4 Lysozyme

Antibacterial bionanostructures were produced from cellulose nanocrystals (CNC) with immobilized lysozyme from hen egg white (HEW) and T4 bacteriophage, respectively. The nanocrystals were prepared from microcrystalline cellulose by ammonium persulfate oxidation with a yield of 68% and having an average size of 250 nm and low polydispersity index. HEW lysozyme (HEWL) and T4 lysozyme (T4L) were immobilized to CNC by different mechanisms including adsorption and covalent coupling to carbodiimide-activated carboxylate groups and to glutaraldehyde-activated aminated CNC (Am-CNC), respectively. The effect of immobilization on the enzymatic activity (both lytic and hydrolytic) and antibacterial activity of the lysozymes was studied using different methods. Am-CNC-lysozyme conjugates retained the highest lytic activity, 86.3% and 78.3% for HEWL and T4L, respectively. They also showed enhanced bactericidal activity with high potency against Gram-positive as well as Gram-negative bacteria in a relatively shorter time as compared to the free enzymes and resulted in extensive cellular damage, as shown by transmission electron microscopy. The enhanced antibacterial activity was correlated with the increase in zeta potential of Am-CNC-lysozyme conjugates. The immobilized lysozyme preparations further exhibited enhanced storage stability at 4 and 22 °C

Kinetic parameters for 3-HPA reduction/1,3-PDO oxidation by purified PduQ-His6 and ADH7-His6.

<p>Kinetic parameters for 3-HPA reduction/1,3-PDO oxidation by purified PduQ-His6 and ADH7-His6.</p

Redox Balance in <i>Lactobacillus reuteri</i> DSM20016: Roles of Iron-Dependent Alcohol Dehydrogenases in Glucose/ Glycerol Metabolism - Fig 6

<p><b>Scheme for double-crossover mutant construction with removal of lreu_1734 gene (A), lreu_0321 gene (B) and lreu_0030 gene (C).</b> Firstly, the gene-specific mutagenesis vector was transformed into cells, after which the target gene was replaced by a <i>lox66-</i>P32-<i>cat-lox71</i> cassette according to homologous recombination. Then, the double-crossover mutant was selected by proper antibiotics, after which the <i>lox66-</i>P32-<i>cat-lox71</i> cassette would be resolved to a single double-mutant <i>lox72</i> site by transient <i>Cre</i> expression from the curable plasmid. Primers used were indicated as black arrowheads (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168107#pone.0168107.s007" target="_blank">S1 Table</a>).</p

NAD(P)⁺ regeneration catalyzed mainly by three enzymes in <i>L</i>. <i>reuteri</i> (Alcohol dehydrogenase (EC1.1.1.1), Lactate dehydrogenase (EC1.1.1.27) and NADH dehydrogenase (EC1.6.99.3)).

<p>ADHs catalyse the NAD(P)⁺-dependent interconversion of aldehydes or ketones and enantiomeric alcoholic compounds. The complete structure of NAD⁺/NADH is indicated by a green H-atom, while the additional phosphate group of NADP⁺/NADPH is indicated in blue.</p

Growth and production characteristics of <i>L</i>. <i>reuteri</i> DSM20016 and its different mutants.

<p>(A) Maximum growth rate; (B) glycerol and glucose consumption and end-product formation in growing cells; (C) 3-HPA, 1,3-PDO and 3-HP production from 300 mM glycerol using resting cells; and (D) ratio of 3-HPA/1,3-PDO and 3-HPA/1,3-PDO during glycerol conversion by the resting cells. Mutant cells LCH010, LCH011, LCH012 involve deletions of PduQ, ADH6 and ADH7, respectively.</p