Purification and characterization of ?-xylosidase from Streptomyces sp. CH7 and its gene sequence analysis

Abstract

A thermostable β-xylosidase was extracted and purified from Streptomyces sp. CH7 mycelium. The apparent molecular weight of the native enzyme estimated by gel filtration was around 173 and 87 kDa for the two subunits estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Its optimal pH and temperature were 6.5 and 55 °C, respectively. The enzyme was stable at pH 6–9 and at 50 °C after 30 min. The K m values for p-nitrophenyl-β--xylopyranoside and o-nitrophenyl-β--xylopyranoside were 0.56 and 0.94 mM with the V m values of 26.3 and 6.6 U/mg protein, respectively. The enzyme was inhibited strongly by Hg2+, Fe2+, Cu2+ and Zn2+. It was inhibited by xylose competitively for p-nitrophenyl-β--xylopyranoside with the K i value of 40 mM. Characterization of the nucleotide sequence of pCH7-1 carrying the β-xylosidase gene from Streptomyces sp. CH7 revealed 3 open reading frames (ORF). The first truncated ORF, bxlI, encodes a putative ABC-type sugar transport system, permease component. The second ORF, bxl2, encodes β-xylosidase, while the third truncated ORF, bxl3, encodes a putative oxidoreductase. The deduced 791 amino acid sequence of Bxl2 showed 84, 71 and 66% identity to those of Streptomyces coelicolor, Streptomyces lividansand Streptomyces thermoviolaceus, respectively. The calculated molecular mass of the deduced amino acid sequence revealed close similarity to that of the purified enzyme.