Molecular Analysis of a Cellulase/xylanase gene,celD, From the Ruminal Bacterium Ruminococcus Flavefaciens Fd-1

A genomic library of Ruminococcus flavefaciens FD-1 DNA was constructed using the Escherichia coli bacteriophage $\lambda$ vector $\lambda$DASH. A recombinant phage exhibiting activity against both Ostazin brilliant red-hydroxyethyl cellulose and carboxymethyl cellulose (CMC) was isolated. This clone (designated FD1-1) was further analyzed by restriction endonuclease mapping and Southern blot analysis. Substrate specificity data show that the cloned gene encodes both endoglucanase and endoxylanase activities. CMC and xylan zymograms of protein produced by FD1-1 and separated by non-denaturing PAGE suggest that the endoglucanase and endoxylanase activities reside on the same polypeptide. Transposon mutagenesis using Tn5supF localized the celD gene within the insert of FD1-1 and supported the zymogram results that the cellulase and xylanase activities were located on a single polypeptide. DNA sequence analysis of celD revealed an open reading frame of 1086 nucleotides encoding a 362 amino acid polypeptide with a molecular mass of 46,329 daltons. The CelD polypeptide had common features with other cellulases e.g. a putative catalytic domain, hydroxyamino acid rich domain, a putative cellulose binding domain, and repeated amino acid sequences near the C-terminus. In addition, CelD showed amino acid sequence similarity with other Ruminococcus cellulases.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Animal Sciences

Grantor

University of Illinois at Urbana-Champaign

Date dc:date

10000-01-01

Author dc:creator

• Howard, Gary Thomas

Contributors dc:contributor

• White, Bryan A.

• Biology, Molecular
• Biology, Microbiology

Identifier

(UMI)AAI9305555

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/72214