Cloning and characterization of the β-xylosidase from Dictyoglomus turgidum for high efficient biotransformation of 10-deacetyl-7-xylosltaxol.

Abstract

With the aim of finding an extracellular biocatalyst that can efficiently remove the C-7 xylose group from 10-deacetyl-7-xylosltaxol, a Dictyoglomus turgidum β-xylosidase was cloned and expressed in Escherichia coli BL21 (DE3). The molecular mass of purified Dt-Xyl3 was approximately 84 kDa. The recombinant Dt-Xyl3 was most active at pH 5.0 and 75 °C, retaining 88% activity at 65 °C for 1 h, and displaying excellent stability over pH 4.0-7.5 for 24 h. In terms of kinetic parameters, the Km and Vmax values for pNPX were 0.8316 mM and 5.0178 μmol/mL·min, respectively. Moreover, Dt-Xyl3 was activated by Mn2+ and Ba2+ and inhibited by Cu2+, Ni+ and Al3+. In particular, it displayed high tolerance to salts with 60.8% activity in 20% (w/v) NaCl. Ethanol and methanol at 5-15% showed little effect on the enzymatic activity. Dt-Xyl3 demonstrated multifunctional activities followed by pNPX, pNPAraf and pNPG and had a high selectivity for cleaving the outer xylose moieties of 10-deacetyl-7-xylosltaxol with Kcat/Km 110.87 s-1/mM, which produced 10-deacetyl-taxol to semi-synthesize paclitaxel. Under the optimized conditions (60 °C, pH 4.5, enzyme dosage of 0.5 U/mL), 1 g of 10-deacetyl-7-xylosltaxol was transformed to its corresponding aglycone 10-deacetyl-taxol within 30 min, with a molar conversion of 98%. This is the first report that Dictyoglomus turgidum can produce extracellular GH3 β-xylosidase with highly specific activity for 10-deacetyl-7-xylosltaxol biotransformation, thus leading to the application of β-xylosidase Dt-Xyl3 as a biocatalyst in biopharmaceutics.