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United States Department of Agriculture

Agricultural Research Service

Research Project: EVOLUTIONARY ENZYMES AND SEPARATION PROCESSES FOR IMPROVED BIOREFINING OF CROPS AND RESIDUES

Location: Bioproduct Chemistry and Engineering Research

Title: A novel xyloglucan-specific endo-beta-1,4-glucanase: biochemical properties and inhibition studies

Authors
item WONG, DOMINIC
item CHAN, VICTOR
item Mccormack, Amanda
item BATT-THRONE, SARAH

Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 12, 2009
Publication Date: June 16, 2010
Citation: Wong, D., Chan, V.J., Mccormack, A.A., Batt Throne, S.B. 2010. A novel xyloglucan-specific endo-beta-1,4-glucanase: biochemical properties and inhibition studies. Applied Microbiology and Biotechnology. 86:1463-1471.

Interpretive Summary: Xyloglucan is one of the major matrix polysaccharides in plant cell wall where it associates with cellulose, providing molecular crosslinks between adjacent cellulose microfibrils, resulting in a three-dimensional cellulose-xyloglucan network. The breakdown of these crosslinks will enhance the digestibility of the cellulose and enzyme conversion to fermentable sugars. This report describes the isolation and cloning of a novel gene of microbial source, and the production of the enzyme that could cleave xyloglucans internally to small fragments, thus release the entangled cellulose microfibrils. This enzyme could contribute to unfolding the cell wall matrix, making it more accessible for complete breakdown of biomass in the conversion to biofuel and bioproducts.

Technical Abstract: A novel xyloglucan-specific endo-β-1,4-glucanase gene (xeg5A) was isolated, cloned, and expressed in E. coli. The enzyme XEG5A consisted of a C-terminal catalytic domain, and a N-terminal sequence of ~90 amino acid residues with unknown function. The catalytic domain assumed an (α/β)8 fold typical of glycoside hydrolase (GH) family 5, with the two catalytic residues GH Glu240 and Glu362 located on opposite sides of the surface groove of the molecule. The recombinant enzyme showed high specificity towards tamarind xyloglucan and one-half of the activity towards xyloglucan oligosaccharide (HDP-XGO). The end products of hydrolysis consisted of three major oligosaccharides, XXXG, XXLG/XLXG, and XLLG. The hydrolysis of tamarind xyloglucan followed the Michaelis-Menten equation, yielding Km and Vmax of 3.61±0.23 mg/ml and 0.30±0.01 mg/ml/min, respectively. However, the hydrolysis of HDP-XGO showed a decrease in the rate at high concentrations suggesting appearance of excess substrate inhibition. The addition of XXXG resulted in linear noncompetitive inhibition on the hydrolysis of tamarind xyloglucan giving a Ki of 1.46±0.13 mM in addition to the Km, app and Vmax, app of 8.08±1.07 mg/ml and 0.49±0.04 mg/ml/min. The enzyme was devoid of transglycosylase activities.

Last Modified: 8/27/2014
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