Fungal glucuronoyl esterases: Genome mining based enzyme discovery and biochemical characterization

Abstract

4-O-Methyl-d-glucuronic acid (MeGlcA) is a side-residue of glucuronoarabinoxylan and can form ester linkages to lignin, contributing significantly to the strength and rigidity of the plant cell wall. Glucuronoyl esterases (4-O-methyl-glucuronoyl methylesterases, GEs) can cleave this ester bond, and therefore may play a significant role as auxiliary enzymes in biomass saccharification for the production of biofuels and biochemicals. GEs belong to a relatively new family of carbohydrate esterases (CE15) in the CAZy database (www.cazy.org), and so far around ten fungal GEs have been characterized. To explore additional GE enzymes, we used a genome mining strategy. BLAST analysis with characterized GEs against approximately 250 publicly accessible fungal genomes identified more than 150 putative fungal GEs, which were classified into eight phylogenetic sub-groups. To validate the genome mining strategy, 21 selected GEs from both ascomycete and basidiomycete fungi were heterologously produced in Pichia pastoris. Of these enzymes, 18 were active against benzyl d-glucuronate demonstrating the suitability of our genome mining strategy for enzyme discovery.

The structural basis of fungal glucuronoyl esterase activity on natural substrates

“…3e). CE15-A comprises mostly bacterial enzymes, but also the fungal subgroup previously referred to as PPR8 6 or G1 27 . In CE15-B, the catalytic acid is located after β7 and is always a glutamic acid.…”

“…CE15-B comprises mostly fungal enzymes, including CuGE, CiP2, and StGE2, but also enzymes of bacterial origin. Variations in the identity and position of the catalytic acid among members of CE15 have been discussed previously 6,7,[13][14][15]18,27,28 . However, with our direct reference to the α/β-hydrolase superfamily we have been able to obtain a clear division of CE15 enzymes into two subgroups with fundamentally different properties encoded in their structural arrangements.…”

Investigation of a thermostable multi-domain xylanase-glucuronoyl esterase enzyme from Caldicellulosiruptor kristjanssonii incorporating multiple carbohydrate-binding modules

“…These are grouped into classes and families in the CAZy database (http://www.cazy.org; [14]) based on their amino acid sequences, which consequently govern their structure and activity. Enzymatic degradation of xylan is typically achieved by xylanases found in glycoside hydrolase families 10 (GH10) and 11 (GH11), although xylanase activity has also been observed for members of families 5,7,8,30,43,98, and 141 [3,[14][15][16][17][18]. Xylanases exist in two major categories: endo-acting xylanases, which randomly cleave backbone linkages in xylan, releasing xylooligosaccharides of varying lengths, and exo-acting xylanases, which remove d-xylose residues from the non-reducing ends of poly-or oligosaccharides [19].…”

“…While extensive data on CE1 enzymes do not exist in the literature, structural and mechanistic insights into these enzymes have however been available for around two decades [28]. Conversely, glucuronoyl esterase (GE) enzymes, known to be able to cleave the ester linkages between GlcA moieties in xylan and the aromatic alcohols on lignin, have since their discovery only recently begun to receive more attention [29][30][31][32][33][34][35].…”

A De Novo Designed Esterase with p-Nitrophenyl Acetate Hydrolysis Activity

“…Esterases are commonly used in the food, pharmaceutical, agricultural and chemical industries. The methods for obtaining new esterases mainly include mining existing natural biological resources such as extreme environment organisms, isolated bacterial genomes, and uncultured metagenomes [ 1 , 2 ]. However, esterases in nature are usually not suitable for the application in harsh environments or newly-emerged reactions.…”