Molecular studies on clostridium thermocellum xylanase gene

Abstract

A gene for encoding xylanase was cloned from Clostridium thermocellum ATCC 27405 in Escherichia coli and the nucleotide sequence of the gene was determined. A C. thermocellum chromosomal DNA fragment which was expected to contain the xylanase gene was previously inserted in pUC19 and constructed a new plasmid pKM29. However, we have found later that the chromosomal insert in pKM29 was lacking part of the xylanase gene including the translational stop codon. Therefore, re-isolation of an entire xylanase gene from C. thermocellum chromosome was performed using pUC19 as a vector followed by screenings for $MUC^+CMC^{-}_pNPG^$ phenotypes. The new plasmid obtained was designated pCX64. The xylanase gene, designated xynX, contained in pCX64 was analyzed for a complete nucleotide sequence using dideoxy chain termination method. The xynX gene composed of an open reading frame comprising 3, 261 bp started with a ATG and stopped with a TAA codon. The molecular weight of protein estimated from the DNA sequence agreed with that of protein produced in Escherichia coli. The A+T content of C. thermocellum DNA was comparatively high, and the 1.6 kb fragment from the C-terminus of xynX gene was not essential for enzyme activity. No typical promoter sequences could be identified in $5^\prime$-noncoding region by sequence homology, and the sequence of ribosome binding site was also found 7 bp upstream from the start codon as the 5-base sequence -GGAGG- complementary with the $3^\prime$-end of Bacillus subtilis 16S rRNA. At the $3^\prime$-noncoding region, there was a sequence having a feature resembling E. coli $\rho$-independent transcription terminator. The calculated free energy of formation for this structure was -15.0 kcal/mol. The xynX encoded enzyme, xylanase X, was highly active on xylan and exhibited activity towards CMC and MUC. The xylanase X hydrolyzed xylan to xylooligosaccharides but showed no activity on crystalline cellulose. The xylanase X behaved as an endoxyl...