Identifying a robust alcohol dehydrogenase for developing one-pot chemobiocatalytic route toward 2,5-bis(hydroxymethyl)furan from glucose

Recently, biomass valorization has attracted great interest. In this work, we present a robust alcohol dehydrogenase from Rhodocyclaceae bacterium (RbADH) for the reduction of 5-hydroxymethylfurfural (HMF), a top valueadded biobased platform chemical, into 2,5-bis(hydroxymethyl)furan (BHMF). The optimal pH and temperature for its activity are pH 7 and 45 degrees C, respectively. Apart from high activities toward a number of aromatic aldehydes (up to 650 U/mg), the enzyme showed high tolerance toward both organic solvents and metal ions. The enzyme displayed good catalytic efficiency (kcat/KM, 140 mM _1 s _ 1 ) toward HMF, with an inhibitory constant (Ki) of 15.3 mM. Recombinant Escherichia coli co-expressing Rb ADH and glucose dehydrogenase was capable of converting HMF of up to 600 mM, providing approximately 71 g/L of BHMF. The space-time yield of up to 3.6 g/ L h was obtained at 500 mM substrate loading. One-pot chemobiocatalytic conversion of inexpensive glucose into BHMF was implemented by sequential chemical isomerization, dehydration and biocatalytic reduction. In preparative-scale synthesis, BHMF was obtained in isolated yields up to 34 % (relative to glucose). The findings of this work may expand the enzyme toolbox and pave the way for sustainable BHMF production.