The natural catalytic function of Cu GE glucuronoyl esterase in hydrolysis of genuine lignin-carbohydrate complexes from birch

Abstract

Background: Glucuronoyl esterases belong to carbohydrate esterase family 15 and catalyze de-esterification. Their natural function is presumed to be cleavage of ester linkages in lignin-carbohydrate complexes particularly those linking lignin and glucuronoyl residues in xylans in hardwood.

Results: Here, we show for the first time a detailed product profile of aldouronic acids released from birchwood lignin by a glucuronoyl esterase from the white-rot fungus Cerrena unicolor (CuGE). CuGE releases substrate for GH10 endo-xylanase which results in significantly increased product release compared to the action of endo-xylanase alone. CuGE also releases neutral xylo-oligosaccharides that can be ascribed to the enzymes feruloyl esterase side activity as demonstrated by release of ferulic acid from insoluble wheat arabinoxylan.

Conclusion: The data verify the enzyme's unique ability to catalyze removal of all glucuronoxylan associated with lignin and we propose that this is a direct result of enzymatic cleavage of the ester bonds connecting glucuronoxylan to lignin via 4-O-methyl glucuronoyl-ester linkages. This function appears important for the fungal organism's ability to effectively utilize all available carbohydrates in lignocellulosic substrates. In bioprocess perspectives, this enzyme is a clear candidate for polishing lignin for residual carbohydrates to achieve pure, native lignin fractions after minimal pretreatment.

Keywords: Aldouronic acids; CE15; Glucuronoxylan; Glucuronoyl esterases; LCC; Lignin.

Fig. 1

LC–MS chromatograms of enzymatically released sodium adduct of aldouronic acids. Charged product profiles generated from treatment of LRP with either CuGE, GH10 endo-xylanase or a combination of the two (presented on the same intensity scale and displayed with an offset between enzyme combinations). The product profiles show a mixture of acetylated aldouronic acids containing 4-O-methyl-glucuronosyl (MeGlcA) ranging from DP 3 to DP 5. Each product is assigned with a number and overall compound composition according to the molecular mass. Several compound masses gave rise to several peaks, i.e., compound (5) indicating structural isomers as a result of differences in substitution pattern. The zoom in the upper right corner shows an enlargement of the product profile resulting from treatment with CuGE alone

Fig. 2

HPAEC-PAD chromatograms of enzyme reaction products and LRP alkali. Comparison of enzyme reaction products released from LRP by CuGE, GH10 endo-xylanase or both from reactions with 5 mg/mL substrate. The chromatograms show a trace of neutral xylo-oligosaccharides from DP1 to DP6 (black) and the NaOH extracted and precipitated glucuronoxylan fraction from LRP as it appears before enzymatic hydrolysis (light blue, LRP alkali, from 37.5 mg/mL substrate). The order of elution on HPAEC is so that neutral components elute first with increasing DP, whereas charged components are retained longer and in this case start to elute after approximately 15 min

Fig. 3

Products released from hydrolysis of LRP by GH10 endo-xylanase and CuGE. a Total amounts of aldotriuronic acid (MeGlcAXyl₂), aldotetrauronic acid (MeGlcAXyl₃), and aldopentauronic acid (MeGlcAXyl₄) released by GH10 endo-xylanase and CuGE on LRP quantified relative to reduced aldotetrauronic acid by LC–MS. The theoretical sum of products released by GH10 and CuGE together is calculated as a sum of the products released by the individual enzymes. Dotted lines indicate the level of the theoretical sum on the actual observed release of products. Total amounts of MeGlcA in molar equivalents originating from the aldouronic acids are represented as a scatter with black markers on the secondary axis. The total MeGlcA concentration in the lignin-rich precipitate is illustrated by an orange horizontal line on the secondary axis (for details on quantification see Additional file 2). b Total amounts of xylose and major xylo-oligosaccharides (DP2-DP4) released by GH10 endo-xylanase and CuGE on LRP. Xylose concentration quantified by HPAEC-PAD and xylo-oligos (DP2-DP4) quantified on LC–MS. Black scatter markers referring to the secondary axis represent the calculated total release in xylose mole equivalents originating from aldouronic acids, xylose, and xylo-oligos. Total amount of xylose in LRP after acid hydrolysis is indicated by an orange horizontal line. All quantifications are performed in triplicate and depicted with standard deviations. Calculated MeGlcA and xylose equivalents are depicted with pooled standard deviations