Nucleotide Sequence of the Clostridium stercorarium xylA Gene Encoding a Bifunctional Protein with β-D-Xylosidase and α-L-Arabinofuranosidase Activities, and Properties of the Translated Product

Abstract

The nucleotides of the β-xylosidase (xylA) gene from Clostridium stercorarium were sequenced. A single open reading frame of 473 codons specifying the subunit (MW 53, 340) of xylosidase was identified. The N-terminal amino acid sequence and molecular weight estimated by SDS-polyacrylamide gel electrophoresis of the purified enzyme were quite in agreement with those deduced from the nucleotide sequence. Analysis of the enzyme by gel filtration on an HPLC column gave a molecular weight of 220, 000, suggesting that the native enzyme is a tetramer composed of 4 identical subunits. The pH optimum was 7.0 and quite stable over the pH range of 5 to 10 at 4°C. The optimum temperature was 65°C. V_m was estimated to be 5.9 nmol/min/μg for p-nitrophenyl-β-D-xylopyranoside and 16.7 nmol/min/μg for p-nitrophenyl-α-L-arabinofuranoside, while K_m was estimated to be 2.5 mM for p-nitrophenyl-β-D-xylopyranoside and 17.6 mM for p-nitrophenyl-α-L-arabinofuranoside.