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United States Department of Agriculture

Agricultural Research Service

Research Project: Biorefining Processes

Location: Bioproduct Chemistry and Engineering Research

Title: Isolation and characterization of a novel GH67 a-glucuronidase from a mixed culture

Authors
item Lee, Charles
item Wagschal, Kurt
item Ruiping, Li -
item Robertson, George
item Kibblewhite, Rena
item Orts, William

Submitted to: Journal of Industrial Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 20, 2012
Publication Date: April 24, 2012
Citation: Lee, C.C., Wagschal, K.C., Ruiping, L., Robertson, G.H., Kibblewhite, R.E., Orts, W.J. 2012. Isolation and characterization of a novel GH67 a-glucuronidase from a mixed culture. Journal of Industrial Microbiology and Biotechnology. 39:1245-1251. DOI:10.1007/s10295-012-1128-7.

Interpretive Summary: For wood or straw to be converted to ethanol by fermentation, its complex carbohydrates must be broken down to simple sugars. Hemicelluloses represent a large reservoir of plant cell wall carbohydrates that can and should be utilized for renewable products but they have proven to be difficult to isolate. Breakdown of hemicellulose into simple sugars is inhibited by its various chemical substituents. One main carbohydrate in hemicellulose, glucuronic acid, is removed by the enzyme a-glucuronidase. In this work, the gene (deg75-AG) that encodes for this enzyme was isolated from a culture of mixed compost microorganisms and this gene was subcloned into a yeast. Subcloning proved successful and the enzyme was overexpressed, well beyond the typical output of this enzyme, and the enzyme was characterized. The enzyme had good activity for breaking down hemicellulose, with optimum activity at 45oC and pH optimum of 7–8. When birchwood was used as the source of hemicellulose, the addition of this enzyme more than doubled the rate of breakdown to xylanase alone.

Technical Abstract: Hemicelluloses represent a large reservoir of carbohydrates that can be utilized for renewable products. Hydrolysis of hemicellulose into simple sugars is inhibited by its various chemical substituents. The glucuronic acid substituent is removed by the enzyme a-glucuronidase. A gene (deg75-AG) encoding a putative a-glucuronidase enzyme was isolated from a culture of mixed compost microorganisms. The gene was subcloned into a prokaryotic vector, and the enzyme was overexpressed and biochemically characterized. The DEG75-AG enzyme had optimum activity at 45 °C. Unlike other a-glucuronidases, the DEG75-AG had a more basic pH optimum of 7–8. When birchwood xylan was used as substrate, the addition of DEG75-AG increased hydrolysis twofold relative to xylanase alone.

Last Modified: 7/25/2014
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