EVOLUTIONARY ENZYMES AND SEPARATION PROCESSES FOR IMPROVED BIOREFINING OF CROPS AND RESIDUES
Location: Bioproduct Chemistry and Engineering Research
Title: Cloning and characterization of an exo-xylglucanase from rumenal microbial metagenome
Submitted to: Protein and Peptide Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 21, 2009
Publication Date: March 1, 2010
Citation: Wong, D., Chan, V.J., Mccormack, A.A., Batt Throne, S.B. 2010. Cloning and characterization of an exo-xylglucanase from rumenal microbial metagenome. Protein and Peptide Letters. 17:803-808.
Interpretive Summary: In the plant cell wall, xyloglucans are a major carbohydrate polymer that intimately associates with cellulose forming a complex network. This type of crosslink interactions confers rigidity and extensibility to the cell wall. The breakdown of xyloglucans using enzymes would liberate the cellulose fibrils from the the crosslinking network and decrease the recalcitrance of the plant biomass materials. This report describes the isolation, expression, and characterization of an enzyme, exo-xyloglucanase, which can efficiently cleave xyloglucan polymers to small fragments. This enzymatic action would potentially disintegrate the xyloglucan-cellulose network to enhance the digestibility of the plant cell wall.
A novel exo-glucanase gene (xeg5B) was isolated from a rumenal microbial metagenome, cloned, and expressed in E. coli. The 1548 bp gene coded for a protein of 516 amino acids, which assumed an (ƒÑ/ƒÒ)8 fold typical of glycoside hydrolase (GH) family 5. The protein molecule consisted of a loop segment blocking one end of the active site, which potentially provided the enzyme with exo-acting property. The recombinant enzyme showed exclusive specificity towards only xyloglucan and oligoxyloglucan substrates with no detectable activity on unsubstituted linear glucans, CMC, laminarin, and lichenan. The major end products of exhaustive hydrolysis were XX (tetrasaccharide) and XG (trisaccharide). The hydrolysis of tamarind xyloglucan followed the Michaelis-Menten kinetics, yielding Km and Vmax of 2.12¡Ó0.13 mg/ml and 0.17¡Ó0.01 mg/ml/min (37oC, pH 6.0), respectively.