A glucuronoyl esterase from Acremonium alcalophilum cleaves native lignin‐carbohydrate ester bonds

Abstract

Edited by Ulf-Ingo Fl€ uggeThe Glucuronoyl esterases (GE) have been proposed to target lignin-carbohydrate (LC) ester bonds between lignin moieties and glucuronic acid side groups of xylan, but to date, no direct observations of enzymatic cleavage on native LC ester bonds have been demonstrated. In the present investigation, LCC fractions from spruce and birch were treated with a recombinantly produced GE originating from Acremonium alcalophilum (AaGE1). A combination of size exclusion chromatography and 31 P NMR analyses of phosphitylated LCC samples, before and after AaGE1 treatment provided the first evidence for cleavage of the LC ester linkages existing in wood.Keywords: 31 P NMR; carbohydrate esterase; lignin-carbohydrate complexes; size exclusion chromatography; spruce and birch An obstacle to intact and efficient extraction of the wood polymers cellulose, hemicelluloses and lignin may be the proposed chemical linkages connecting lignin with hemicelluloses, the so-called lignin-carbohydrate complexes (LCCs). In a lignocellulose-based biorefinery context it is therefore of interest to find mild methods, for example, enzymatic treatment, that specifically would break these lignin-carbohydrate (LC) linkages. Three types of existing covalent LC bonds have been suggested, namely benzyl ether-, ester-and phenyl glycosidic linkages [1]. The first described and most studied LC ester bond is the benzyl a-ester bond [2]. However, several more recent studies have also shown the presence of c-esters [3,4], suggested to arise due to uronosyl migrations of the linkage from the a-to the c-position [5,6].It has been proposed that glucuronoyl esterases (GE), members of the recently discovered carbohydrate esterase 15 family (CE15), could be used to target the LC ester bond between aliphatic alcohols in lignin and the 4-O-methyl-D-glucuronic acid residues of glucuronoxylans ( Fig. 1) [7,8].The first characterized GE was produced by the wood-rotting fungus Schizophyllum commune [7] and at the present time nine GEs have been characterized for their activity and kinetics on different model substrates [7][8][9][10][11][12][13][14][15][16][17][18][19][20].Cleavage of model substrates mimicking LC ester bonds has been investigated for a wide range of esters. Studies have covered simple glucuronic acid (GlcA) esters with the 'lignin moiety' being single arylalkyl units at the c-position [8,10] or at the a-position [14].

Characterisation of three fungal glucuronoyl esterases on glucuronic acid ester model compounds

“…Of those, the d -benzyl glucuronic acid ester (R alcohol  = benzyl; R 1 , R glycosidic  = H) was reported to be suitable for quantitative estimation of GE activity using HPLC, while allylGlcA (R alcohol  = allyl; R 1 , R glycosidic  = H) and MeGlcA (R alcohol  = methyl; R 1 , R glycosidic  = H) have only been used in TLC-based assays (Sunner et al 2015; De Santi et al 2016). In a recent study, Arnling Bååth et al also demonstrated GE action on a natural substrate by using native lignin-carbohydrate ester bonds from spruce and birch (Arnling Bååth et al 2016).

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“…It was recently demonstrated that GEs can cleave the ester bond between lignin and glucuronoxylan (Arnling Bååth et al 2016), pointing to the potential of this enzyme to aid in targeted decomposition of biomass. As GEs enable selective cleavage of alkali-labile bonds at acidic pH values, GEs would also allow biomass degradation at a lower pH where other alkali-labile structures can be retained.…”

Microbial Glucuronoyl Esterases: 10 Years after Discovery

“…However, the recent evidence that the addition of GEs to saccharification enzyme systems improves sugar yields from pretreated plant biomass (25,30), as well as the observation that GEs are present in commercial enzyme preparations (38), underlines the need to elucidate further the role of GEs in microbial degradation of plant cell walls and other biotechnological potential, particularly in biorefinery and saccharification processes. This is stressed by the recent observation that ligninhemicellulose esters are present in both hardwood and softwood (26). As indicated by patent literature on GEs (30), new opportunities can be foreseen in the area of genetic modification of plants by expression of microbial GE genes.…”

“…A recombinant GE from Acremonium alcalophilum reduced the molecular mass of isolated lignin-carbohydrate complexes from spruce and birch, as shown by size exclusion chromatography (26). Simultaneous analyses of hydroxyl groups before and after GE treatment of subsequently phosphitylated derivatives of the complexes by 31 P-NMR spectroscopy (27) showed an increase in the content of the carboxyl hydroxyl groups as a result of de-esterification of uronic acids (26). The enzymes certainly play an important role in plant cell wall degradation since their addition to a commercial cellulolytic system from Novozymes considerably enhanced the amount of liberated reducing sugars, including both pentoses and hexoses (25).…”

“…Natural sub- strates derived from plant cell wall material, such as suitable lignin-carbohydrate complexes, await their introduction. The isolation, purification, and characterization of such complexes, as well as the synthesis of their analogues, are not easy tasks (4,26). Benzyl glucuronate, which is the only synthetic substrate commercially available, was recently suggested for both qualitative and quantitative GE assays, including a uronic acid dehydrogenasecoupled assay (36).…”