METABOLISM OF 5-HYDROXYMETHYL FURFURAL IN Lentibacillus salarius BPIITR AND ITS EFFECT ON BRANCHED CHAIN AMINO ACIDS BIOSYNTHESIS

Abstract

Branched-chain amino acids (BCAA) isoleucine, leucine and valine, are central molecules involved in a wide variety of pathways, ranging from the synthesis of essential intermediates for physiological stress regulation. Catabolism of BCAA yields amino acid specific ketoacids which further converted to respective branched chain fatty acids. However, the internal pool of these branched-chain amino acids varies depending on the nutritional availability and stress conditions. 5-hydroxymethyl furfural (5-HmF) is a carcinogenic compound, derived from dehydration of hexose sugars found in food and cellulosic biorefinery wastewater. The metabolism of such carcinogens has been studied in the literature in the context of biotransformation. Halophilic microorganisms are found in environment and salt rich food matrices. They possess distinct potential to degrade the recalcitrant and toxic molecules. In present study, a halophilic bacterium Lentibacillus salarius BPIITR was studied to elucidate its ability to degrade toxic 5-HmF and pathways associated with BCAA catabolism. Supplementation of 5-HmF along with glucose as carbon source increased the biomass to 5.22 ± 0.14 mg mL-1, whereas it decreased to 1.89 ± 0.02 mg mL-1 and 2.86 ± 0.22 mg mL-1 with 5-HmF and glucose as sole carbon source, respectively (p<0.05). In presence of 5-HmF, different substrates were studies to explore the potential oxidase enzyme preferred substrate, i.e., 2-methoxy benzyl alcohol (2-MBA), 4-hydroxy benzyl alcohol (4-HBA), and 2,6-dimethoxy phenol (2,6-DMP). Among the studied substrates, 2-methoxy benzyl alcohol exhibited highest selectivity during 5-HmF degradation extracellular activity was measured with different substrates such as 2-methoxy benzyl alcohol, 2-hydroxy benzyl alcohol and 2,6-dimethoxy phenol. During glucose supplementation, 2,6-DMP and 2-MBA activities were 18.7 ± 2.15 U mL-1 and 18.9 ± 0.84 U mL-1, respectively. Supplementation of 5-HmF decreased the affinity towards 2,6-DMP, however, 2-MBA and 2-HBA as substrates enzyme activities were increased to 49.4 ± 3.16 7 U mL-1 and 39.4 ± 1.33 U mL-1. Similar trend was observed in presence of 5-HmF as sole carbon source, however, 2-MBA yielded activity of 86.2 ± 3.84 U mL-1. The degraded products of 5-HmF were observed using HPLC with PDA detector. Further BCAA variation in presence of 5-HmF were identified through amino acids alkylation strategy and correlated using heatmap. The correlation exhibited strong positive correlation between isoleucine and threonine. In principal component analysis the strong changes in amino acids were detected as a cluster in presence of 5-HmF as sole carbon source. Threonine deaminase, aceto-hydroxy acid synthase and ketoacid decarboxylase activities were measured to elucidate the preferred synthesis of BCAA during exposure to 5-HmF. Threonine deaminase activity increased to 1051.5 ± 171.11 U ng-1 protein in presence of 5 HmF, whereas it decreased to 558.2 ± 85.4 U ng-1 protein during supplementation with glucose. Isoleucine mediated inhibition of threonine deaminase was found to be less effective during 5-HmF exposure. For instance, at 25 µM isoleucine concentration, threonine deaminase activity completely hindered in glucose fed control medium, whereas supplementation of 5-HmF sustained the threonine deaminase activity up to 34.42 ± 1.6 %, and 56.01 ± 3.0 %, respectively. Pyruvate assisted BCAA synthesis was elucidated by measuring aceto-hydroxy acid synthase (AHAS) activity. In glucose as sole carbon source, AHAS activity found high i.e., 6119.2 ± 532.5 U mg-1 of protein, whereas it decreased to 3818.8 ± 180.9 U mg-1 of protein and 2732.6 ± 193.2 U mg-1 of protein during supplementation of 5-HmF with glucose and as sole carbon source, respectively. The preferential decarboxylation of ketoacids were in the order of isoleucine > valine > leucine, which suggests the essential role of isoleucine in presence of toxic compounds. Highlighting the ability of halophilic bacterium to degrade the toxic 5-HmF, further studies will be required to elucidate the physiological role of BCAA during toxic environment exposure.