The pie chart of the distribution of PDOR’ activities and identified mutants.

<p>The pie chart of the distribution of PDOR’ activities and identified mutants.</p

Directed Evolution and Resolution Mechanism of 1, 3-Propanediol Oxidoreductase from <i>Klebsiella pneumoniae</i> toward Higher Activity by Error-Prone PCR and Bioinformatics

<div><p>1, 3-propanediol oxidoreductase (PDOR) is a key enzyme in glycerol bioconversion to 1,3-propanediol (1, 3-PD) which is a valuable chemical and one of the six new petrochemical products. We used error-prone PCR and activity screening to identify mutants of <i>Klebsiella pneumoniae</i> (<i>K</i>. <i>pneumoniae</i>) PDOR with improved activity. The activity of one of the identified mutants, PDOR’-24, which includes a single mutation, A199S, was 48 U/mg, 4.9 times that of the wild-type enzyme. Molecular docking was performed to analyze the identified mutants; and amino acids S103, H271, N366, D106, N262 and D364 were predicted to bond with NADH. The origins of the improved activity of PDOR’-24, as well as three other mutants were analyzed by simulating the interaction mechanism of the mutants with the substrate and coenzyme, respectively. This research provides useful information about the use of safranine O plate screening for the directed evolution of oxidoreductases, identifies interesting sites for improving PDOR activity, and demonstrates the utility of using molecular docking to analyze the interaction mechanism of the mutants with the substrate and coenzyme, respectively.</p></div

Bioconversion of glycerol by fused glycerol dehydrogenase and NADH oxidase coupled with NADH regeneration.

<p>Bioconversion of glycerol by fused glycerol dehydrogenase and NADH oxidase coupled with NADH regeneration.</p

Optimal induce time of the T01-08, opt-<i>nox</i> and the wild type.

<p>(a)Comparison of the specific activity of the T01-08 and the wild type with different induced time (1–8h). (b) Optimal inducing time of the opt-<i>nox</i>.</p

Specific activities of the opt-<i>nox</i> in the process of purification.

<p>Specific activities of the opt-<i>nox</i> in the process of purification.</p

Determination of kinetic parameters of GDH-NOX.

<p>(a) Glycerol; (b) NADH. Experiment condition: glycerol concentration (0.01, 0.125, 0.014, 0.025, 0.05M), NADH concentration (20, 40, 60, 100, 200 μM). pH 7.0, 37°C.</p

Sequences of the third to sixth codons and the Nox activities.

<p>Note: Mutation sites was underlined.</p><p>Sequences of the third to sixth codons and the Nox activities.</p

Characterization of the NOX and SDS-PAGE analysis of the purified NOX.

<p>(a) 10% SDS-PAGE analysis of the purification NOX. Lane M: protein marker; Lane 1: purified NOX with His-tag; Lane 2: recombinant bacterium (harboring pET-32a-<i>nox</i>) induced by IPTG. (b) Optimal pH for NOX. (c) The optimal inducing time of NOX.</p

Identification of evolved mutants using the high throughput safranin O plates screening method.

<p>An example of an active and an inactive clone are shown.</p

The bonding model of mutated PDOR’s with NADH or 3-HPA.

<p>A: before the NADH and 3-HPA bonding of mutated PDOR; B: bonding of mutated PDOR and 3-HPA; C: bonding of mutated PDOR and NADH; D: bonding of mutated PDOR, NADH and 3-HPA.</p