Screening of a Xylanase Clone from a Fosmid Library of Rumen Microbiota in Hu Sheep

Abstract

The glycosyl hydrolase family 11, which is responsible for carbohydrate metabolism, was identified in the open reading frame (ORF) 6 of a xylanase positive clone from a fosmid library of rumen microbiota of Hu sheep. A BLASTP search of GenBank revealed that ORF6 encoded a 355-amino acid putative endoxylanase, having 61% similarity (e⁻⁷³) to endo-1,4-β-xylanase of Fibrobacter succinogenes S85 (YP_003250510.1). Predicted with the SWISS-MODEL, there were two separate β-sandwich clusters linked with a high serine containing linker in ORF6. The N-terminal β-sandwich is a novel endoxylanase of the glycosyl hydrolase family 11 with a specific activity of 1150.00 U/mg. The optimal pH and temperature for this enzyme were shown to be pH 5.0 and 50°C, respectively. The C-terminal helped increase the stability of the xylanase but decreased the activity to some degree. The C-terminal β-sandwich could bind avicel, but no conserved domain could be found. It may be a novel carbohydrate-binding module.

Keywords:

• Carbohydrate binding module
• Fosmid
• Physicochemical characterization
• Rumen
• Xylanase

Acknowledgments

We thank Dr. Stuart E. Denman (CSIRO Livestock Industries) for his technical assistance. This work was supported by a grant from the National Natural Science Foundation of China (No. 30800786).