Functional Cloning and Expression of the Schizophyllum commune Glucuronoyl Esterase Gene and Characterization of the Recombinant Enzyme

The gene encoding Schizophyllum commune glucuronoyl esterase was identified in the scaffold 17 of the genome, containing two introns of 50？bp and 48？bp, with a transcript sequence of 1179？bp. The gene was synthesized and cloned into Pichia pastoris expression vector pGAPZα to achieve constitutive expression and secretion of the recombinant enzyme in soluble active form. The purified protein was 53？kD with glycosylation and had an acidic pI of 3.7. Activity analysis on several uronic acids and their derivatives suggests that the enzyme recognized only esters of 4-O-methyl-D-glucuronic acid derivatives, even with a 4-nitrophenyl aglycon but did not hydrolyze the ester of D-galacturonic acid. The kinetic values were 0.25？mM, 16.3？μM·min？1, and 9.27？s？1 with 4-nitrophenyl 2-O-(methyl 4-O-methyl-α-D-glucopyranosyluronate)-β-D-xylopyranoside as the substrate. 1. Introduction In the current schemes of biomass conversion, pretreatment with enzyme hydrolysis recovers only about 85% of the theoretical yield for the available sugars [1]. Development of a cost-competitive process is hampered by the lack of knowledge on the breakdown of covalent cross-linkages connecting cellulose, hemicellulose, and lignin in plant cell walls. As much as 90% of the lignin in woody plants might be covalently linked to polysaccharides [2]. The types of covalent lignin-carbohydrate linkages have been proposed to include lignin alcohol esters, ethers, and phenyl glycosides [3–5]. The wood-rotting fungus Schizophyllum commune has been shown to produce a glucuronoyl esterase (ScGE), which cleaves substrate mimics of ester bonds between lignin alcohols and glucuronoxylan [6]. Other carbohydrate esterases, acetylxylan esterases, feruloyl esterases, and pectin methylesterases, did not act on these substrates. GE enzymes were subsequently isolated from other source microorganisms, including Hypocrea jecorina, Phanerochaete chrysosporium, and Sporotrichum thermophile [7–9]. In this paper, the putative cDNA gene of glucuronoyl esterase in the genome of the original source microorganism, Schizophyllum commune was identified, synthesized, cloned, and expressed in Pichia pastoris. The recombinant enzyme (rScGE) was purified and its enzyme action characterized on uronic acid substrates and their derivatives. 2. Materials and Methods 2.1. Materials and Strains The pGAPZα-A vector, strain SMD1168, Zeocin, protein extraction reagent, precast gel, protein standards and staining kits were purchased from Invitrogen (San Diego, CA, USA). Gene DNA synthesis was performed by EZBiolab (Carmel, IN, USA), and