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

Ján Hirsch,Amanda A Mccormack,Peter Biely,Dominic W S Wong,Victor J Chan

doi:10.1155/2012/951267

Abstract

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 K m 0.25 mM, V max 16.3 μM·min−1, and k cat 9.27 s−1 with 4-nitrophenyl 2-O-(methyl 4-O-methyl-α-D-glucopyranosyluronate)-β-D-xylopyranoside as the substrate.

Highlights

In the current schemes of biomass conversion, pretreatment with enzyme hydrolysis recovers only about 85% of the theoretical yield for the available sugars [1]
GE enzymes were subsequently isolated from other source microorganisms, including Hypocrea jecorina, Phanerochaete chrysosporium, and Sporotrichum thermophile [7,8,9]
The putative gene sequence for Schizophyllum commune glucuronoyl esterase was found to locate at scaffold 17 of the genome (MW 003315656), containing two introns of 50 bp and 48 bp, with a transcript sequence of 1179 bp

Summary

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. The types of covalent lignin-carbohydrate linkages have been proposed to include lignin alcohol esters, ethers, and phenyl glycosides [3,4,5]. 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]. GE enzymes were subsequently isolated from other source microorganisms, including Hypocrea jecorina, Phanerochaete chrysosporium, and Sporotrichum thermophile [7,8,9]. 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

Methods

Results

Conclusion