EXPLORING ENZYMACIC ROUTES FOR 2,5-FURANDICARBOXYLIC ACID PRODUCTION FROM 5-HYDROXYMETHYL FURFURAL IN Lentibacillus salarius BPIITR

Abstract

5-Hydroxymethyl furfural (5-HmF) is lignocellulosic sugar derived furan aldehyde, considered as the major microbial inhibitor during fermentation and a potential feed stock to produce different furan carboxylic acids and biofuels. In the current study, halophile Lentibacillus salarius BPIITR enzymes were considered for exploring the production of 2,5-furan carboxylic acid from 5-HmF, which is an alternative to non-degradable plastic monomers. The microbial culture was grown in salt medium with supplementation of glucose and glucose with 5-HmF to investigate the growth pattern and enzymes envisioned for 5-HmF degradation. In the medium containing only glucose the highest bacterial growth observed was 3.5 ± 0.02 g/L, whereas supplementation of 5-HmF along with glucose enhanced the bacterial growth to 6.6 ± 0.43 g/L. Furthermore, the major enzymes involved in 5-HmF degradation were dehydrogenase and oxidase, which were monitored spectrophotometrically using 5-HmF along with NADH as co-factor and 4-methoxy benzyl alcohol as substrates, respectively. The dehydrogenase activity in glucose and 5-HmF supplemented medium was 39.4 ± 2.4 and 90.3 ± 3.2 IU/mg protein, respectively. On the other hand, oxidase activity enhanced profoundly with 5-HmF supplementation, i.e., 690.2 ± 32.8 IU/mg protein, whereas it was 81.6 ± 6.16 IU/mg in only glucose containing medium. Furthermore 5-HmF degradation products were identified in presence of dehydrogenase and oxidase using high pressure liquid chromatography. Dehydrogenase oxidized the aldehyde group of 5-HmF to produce 5-hydroxymethyl-2-furancarboxylic acid (HMFCA) as major product, whereas oxidase catalyzed alcohol group of 5-HmF to produce 2,5-diformyl furan (DFF), which further oxidized to 5-formyl-2-furancarboxylic acid (FFCA), followed by further oxidation to 2,5 furandicarboxylic acid (FDCA). Considering the significant enhancement in the oxidase activity, further studies were carried out to depict intermediates effects on 5-HmF degradation and major active or inhibition zone in the degradation pathway. In the presence of 5 mM HMFCA and 5mM FFCA, DFF formation was significantly affected between 0 to 1 hr. In addition, at both 2.5 and 5 mM HMFCA along with 5 mM FFCA, HMFCA was further converted to FFCA. During 2.5 mM DFF and 5 mM FFCA, DFF accumulated up to 3.55 ± 0.11 mM, whereas it further degraded at higher rate to FFCA at 5 mM DFF in the reaction mixture. In conclusion, the oxidase enzyme explored in the L. salarius BPIITR has substrate preferences as a function of intermediates concentration. FFCA was identified as a promising substrate for FDCA production due to shorter reaction steps compared to 5-HmF to FDCA.