Oxidation of 5-hydroxymethylfurfural with a novel aryl alcohol oxidase from Mycobacterium sp. MS1601

Bio-based 5-hydroxymethylfurfural (HMF) serves as an important platform for several chemicals, among which 2,5-furan dicarboxylic acid (FDCA) has attracted considerable interest as a monomer for the production of polyethylene furanoate (PEF), a potential alternative for fossil-based polyethylene terephthalate (PET). This study is based on the HMF oxidizing activity shown by Mycobacterium sp. MS 1601 cells and investigation of the enzyme catalysing the oxidation. The Mycobacterium whole cells oxidized the HMF to FDCA (60% yield) and hydroxymethyl furan carboxylic acid (HMFCA). A gene encoding a novel bacterial aryl alcohol oxidase, hereinafter MycspAAO, was identified in the genome and was cloned and expressed in Escherichia coli Bl21 (DE3). The purified MycspAAO displayed activity against several alcohols and aldehydes; 3,5 dimethoxy benzyl alcohol (veratryl alcohol) was the best substrate among those tested followed by HMF. 5-Hydroxymethylfurfural was converted to 5-formyl-2-furoic acid (FFCA) via diformyl furan (DFF) with optimal activity at pH 8 and 30-40 degrees C. FDCA formation was observed during long reaction time with low HMF concentration. Mutagenesis of several amino acids shaping the active site and evaluation of the variants showed Y444F to have around 3-fold higher k(cat)/K-m and similar to 1.7-fold lower K-m with HMF.

Automated summary

This study demonstrates the oxidizing activity of Mycobacterium sp. MS 1601 cells towards HMF, yielding FDCA and HMFCA. The enzyme responsible for this oxidation, MyccspAAO, was identified and shown to exhibit activity against various alcohols and aldehydes, with veratryl alcohol being the best substrate. HMF can be converted to FFCA via DFF under specific conditions. Mutagenesis experiments showed that the Y444F variant had higher catalytic efficiency and lower K-m with HMF.