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Lignocellulose-Degrading Enzymes: A Biotechnology Platform for Ferulic Acid Production from Agro-Industrial Side Streams.

ABSTRACT: Biorefining by enzymatic hydrolysis (EH) of lignocellulosic waste material due to low costs and affordability has received enormous interest amongst scientists as a potential strategy suitable for the production of bioactive ingredients and chemicals. In this study, a sustainable and eco-friendly approach to extracting bound ferulic acid (FA) was demonstrated using single-step EH by a mixture of lignocellulose-degrading enzymes. For comparative purposes of the efficiency of EH, an online extraction and analysis technique using supercritical fluid extraction-supercritical fluid chromatography-mass spectrometry (SFE-SFC-MS) was performed. The experimental results demonstrated up to 369.3 mg 100 g^-1 FA release from rye bran after 48 h EH with Viscozyme L. The EH of wheat and oat bran with Viscoferm for 48 h resulted in 255.1 and 33.5 mg 100 g^-1 of FA, respectively. The release of FA from bran matrix using supercritical fluid extraction with carbon dioxide and ethanol as a co-solvent (SFE-CO₂-EtOH) delivered up to 464.3 mg 100 g^-1 of FA, though the extractability varied depending on the parameters used. The 10-fold and 30-fold scale-up experiments confirmed the applicability of EH as a bioprocessing method valid for the industrial scale. The highest yield of FA in both scale-up experiments was obtained from rye bran after 48 h of EH with Viscozyme L. In purified extracts, the absence of xylose, arabinose, and glucose as the final degradation products of lignocellulose was proven by high-performance liquid chromatography with refractive index detection (HPLC-RID). Up to 94.0% purity of FA was achieved by solid-phase extraction (SPE) using the polymeric reversed-phase Strata X column and 50% EtOH as the eluent.

SUBMITTER: Radenkovs V

PROVIDER: S-EPMC8701750 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Plant cell walls are composed of cellulose, hemicellulose, and lignin, collectively known as lignocellulose. Microorganisms degrade lignocellulose to liberate sugars to meet metabolic demands. Using a metagenomic sequencing approach, we previously demonstrated that the microbiome of the North American porcupine (Erethizon dorsatum) is replete with genes that could encode lignocellulose-degrading enzymes. Here, we report the identification, synthesis and partial characterization of four novel genes from the porcupine microbiome encoding putative lignocellulose-degrading enzymes: ?-glucosidase, ?-L-arabinofuranosidase, ?-xylosidase, and endo-1,4-?-xylanase. These genes were identified via conserved catalytic domains associated with cellulose- and hemicellulose-degradation. Phylogenetic trees were created for each of these putative enzymes to depict genetic relatedness to known enzymes. Candidate genes were synthesized and cloned into plasmid expression vectors for inducible protein expression and secretion. The putative ?-glucosidase fusion protein was efficiently secreted but did not permit Escherichia coli (E. coli) to use cellobiose as a sole carbon source, nor did the affinity purified enzyme cleave p-Nitrophenyl ?-D-glucopyranoside (p-NPG) substrate in vitro over a range of physiological pH levels (pH 5-7). The putative hemicellulose-degrading ?-xylosidase and ?-L-arabinofuranosidase enzymes also lacked in vitro enzyme activity, but the affinity purified endo-1,4-?-xylanase protein cleaved a 6-chloro-4-methylumbelliferyl xylobioside substrate in acidic and neutral conditions, with maximal activity at pH 7. At this optimal pH, KM, Vmax, and kcat were determined to be 32.005 ± 4.72 ?M, 1.16x10-5 ± 3.55x10-7 M/s, and 94.72 s-1, respectively. Thus, our pipeline enabled successful identification and characterization of a novel hemicellulose-degrading enzyme from the porcupine microbiome. Progress towards the goal of introducing a complete lignocellulose-degradation pathway into E. coli will be accelerated by combining synthetic metagenomic approaches with functional metagenomic library screening, which can identify novel enzymes unrelated to those found in available databases.

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Lignocellulose-Degrading Enzymes: A Biotechnology Platform for Ferulic Acid Production from Agro-Industrial Side Streams.

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