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

Abstract

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°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 ~1.7-fold lower K m with HMF.

Identification of redox activators for continuous reactivation of glyoxal oxidase from Trametes versicolor in a two-enzyme reaction cascade

“…HMF is produced during the pretreatment of lignocellulosic biomass 4 . Several studies have dealt with the development of enzymatic routes for the environmentally friendly and sustainable production of FDCA from HMF 5 – 10 . Fungal enzymes involved in lignin degradation have been thoroughly investigated for this purpose 11 – 13 .…”