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United States Department of Agriculture

Agricultural Research Service

Title: Cloning and characterization of an alpha-glucuronidase from a mixed microbial population

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

Submitted to: Enzyme and Microbial Technology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 15, 2009
Publication Date: December 2, 2009
Citation: Lee, C.C., Kibblewhite, R.E., Wagschal, K.C., Robertson, G.H., Orts, W.J. 2009. Cloning and characterization of an alpha-glucuronidase from a mixed microbial population. Enzyme and Microbial Technology. 155(1-3):314-320.

Interpretive Summary: Hemicellulose comprises a large percentage of the world’s biomass. As such, this material represents a rich source of renewable material for the chemical and fuel industries. One of the main components of hemicellulose is xylan, a polymer of beta-1,4-linked xylose residues. The xylan can be hydrolyzed to simple sugars by the enzymatic action of endoxylanase, which cleaves internal 1,4-linkages to produce short chain xylooligomers, and beta-xylosidase, which removes individual xylose monomers from the non-reducing end of the xylooligomers. The xylan polymer is commonly modified with a substituent known as glucuronic acid. The glucuronic acid must be enzymatically removed by an enzyme known as alpha-glucuronidase. We have cloned the first alpha-glucuronidase gene to ever be isolated from a mixed population of microorganisms. The recombinant enzyme was purified and biochemically characterized. It was found to have an activity pH optimum higher than that of any previously reported alpha-glucuronidases. It was also demonstrated to function synergistically with xylanase in the hydrolysis of xylan substrate.

Technical Abstract: Alpha-Glucuronidase enzymes play an essential role in the full enzymatic hydrolysis of hemicellulose. Up to this point, all genes encoding alpha-glucuronidase enzymes have been cloned from individual, pure culture strains. Using a high-throughput screening strategy, we have isolated the first alpha-glucuronidase-encoding gene (deg75-AG) from a mixed population of 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 45oC. Unlike most other alpha-glucuronidases, the DEG75-AG had a more basic pH optimum of 7 to 8. When birchwood xylan was used as substrate, the addition of DEG75-AG increased hydrolysis 2-fold relative to xylanase alone.

Last Modified: 12/19/2014
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