Electrochemical Hydroxylation of C3\u2013C12n-Alkanes by Recombinant Alkane Hydroxylase (AlkB) and Rubredoxin-2 (AlkG) from Pseudomonas putida GPo1

Yi-Fang Tsai,Jen-Lin Chang,Chun-Wei Chang,Huai-Chun Chuang,Guor-Tzo Wei,Ravirala Ramu,Wen-I Luo,Steve S.-F. Yu,Jyh-Myng Zen

doi:10.1038/s41598-017-08610-w

Abstract

An unprecedented method for the efficient conversion of C3–C12 linear alkanes to their corresponding primary alcohols mediated by the membrane-bound alkane hydroxylase (AlkB) from Pseudomonas putida GPo1 is demonstrated. The X-ray absorption spectroscopy (XAS) studies support that electrons can be transferred from the reduced AlkG (rubredoxin-2, the redox partner of AlkB) to AlkB in a two-phase manner. Based on this observation, an approach for the electrocatalytic conversion from alkanes to alcohols mediated by AlkB using an AlkG immobilized screen-printed carbon electrode (SPCE) is developed. The framework distortion of AlkB–AlkG adduct on SPCE surface might create promiscuity toward gaseous substrates. Hence, small alkanes including propane and n-butane can be accommodated in the hydrophobic pocket of AlkB for C–H bond activation. The proof of concept herein advances the development of artificial C–H bond activation catalysts.

Highlights

Artificial direct oxidation on terminal C–H bond of linear alkanes leading to primary alcohols in a highly selective manner is acknowledged to be challenging[1,2,3,4,5]
The analysis of XANES spectra obtained from AlkGoxidized, AlkGreduced, AlkBoxidized and AlkB–AlkG pair yields the plausible electron transfer pathway employed by AlkB through the transition of AlkBoxidizedAlkGoxidized → AlkBoxidizedAlkGreduced → AlkBreducedAlkGreduced, further facilitating the hydroxylation of n-alkanes (Fig. 1)
This revelation allows the development of a system for artificial n-alkane hydroxylation using AlkG immobilized screen-printed carbon electrode (SPCE), in which AlkT is replaced by the electrode

Summary

Introduction

Artificial direct oxidation on terminal C–H bond of linear alkanes leading to primary alcohols in a highly selective manner is acknowledged to be challenging[1,2,3,4,5]. (formerly known as Pseudomonas oleovorans) found in nature, can regio-selectively introduce molecular oxygen onto the unreactive terminal methyl group of C5−C12 linear alkanes to yield primary alcohols and water in the presence of reducing equivalents (eq 1). Taking advantage of this protein, P. putida GPo1 is able to utilize linear medium-chain length alkanes (C5−C12) as the sole source of carbon and energy[6,7,8,9]. A comparison between soluble and membrane bound non-heme diiron proteins as well as the sequence alignment data among AlkB, SCD1 and XylM are illustrated in Supplementary Information, Tables S1 and S2 to reveal their functional features as well as structural and biochemical characteristics. The metal active-site, the environment around the active-site and the www.nature.com/scientificreports/

Methods

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Conclusion