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Title: CLONING, EXPRESSION, AND SEQUENCING OF AN ARABINOFURANOSIDASE GENE FROM THE RUMINAL ANAEROBE BUTYRIVIBRIO FIBRISOLVENS H17C

Author
item Whitehead, Terence
item Lee, David

Submitted to: Conference on Rumen Function
Publication Type: Abstract Only
Publication Acceptance Date: 11/16/1995
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Improving digestion of xylan in the rumen through genetic manipulation of ruminal microorganisms has been suggested as a means to enhance ruminal utilization of this polysaccharide. Butyrivibrio fibrisolvens is one of the more important ruminal xylan-degrading microorganisms and would be a prime choice for this work as it expresses a full complement of xylanolytic enzymes (xylanase, xylosidase, arabinosidase, acetyl esterase, glucuronidase). As a part of this work, we have cloned a genetic locus from B. fibrisolvens H17c that produces an arabinofuranosidase in Escherichia coli, as initially determined by screening on methylumbelliferyl-arabinofuranoside. The enzyme expressed in E. coli was capable of releasing arabinose from wheat arabinoxylan and oatspelt xylan. Sequencing of the locus indicated that the entire gene had not been cloned on the Sau3A fragment. A second EcoRI locus was cloned which contained the entire gene. DNA sequencing of the locus demonstrated the presence of an open reading frame corresponding to the arabinosidase activity which encodes for a protein of 88,000 molecular weight. Analyses of the derived protein sequence did not result in any significant amino acid similarity with other reported arabinosidases. Attempts will be made to introduce the cloned arabinosidase gene back into B. fibrisolvens H17c using E. coli/B. fibrisolvens shuttle vectors and determine if overexpression of the gene occurs. The effect of overexpression on the ability of this organism to degrade and utilize xylan will also be determined.