Molecular Cloning, Heterogenous Expression And Biochemical Characterization Of A β-xylosidase From Enterobacter Sp. CGMCC2487

Paclitaxel is a secondary metabolite from Taxus species. It is effective to treat several cancers, such as ovarian, breast cancer and so on. The content of paclitaxel is approximate0.02%in yew, however, the content of7-xylosyl-10-deacetyl taxol is more than10folds higher than paclitaxel, but7-xylosyl-10-deacetyl taxol is discarded as waster in the extraction of paclitaxel. Because7-xylosyl-10-deacetyl taxol can be converted to10-deacetyl taxol by hydrolyzing xylosyl at C-7and the latter can be converted to taxol by one step of acylation, exploring the methods to transform7-xylosyl-10-deacetyl taxol to10-deacetyl taxol is economically important.In early experiments we screened a bacterial strain--Enterobacter sp. CGMCC2487which can hydrolyse7-xylose-10-deacetyl taxol to10-deacetyl taxol efficiently. On this study, The β-xylosidase gene from Enterobacter sp. CGMCC2487was cloned by a way of constructing Enterobacter sp. CGMCC2487genomic library. The β-xylosidase gene was cloned into pET-23a vector and pHBM905A vector and the two vestors were transformed into E.coli BL21and Pichia pastoris GS115respectively for expression. Then the expression products were purified by using the High-Affinity Ni-NTA Resin. Enzymatic properties of the β-xylosidase, including xylosidase activity, optimum temperature and temperature stability, optimum pH and pH stability, the impacts of the xylosidase activity by xylose, metal ions and some other reagents, were measured by using4-nitrophenyl-β-D-xylopyranoside (pNPX) as the substrate. The efficiency of hydrolysis of7-xylose-10-deacetyl taxol to10-deacetyl taxol was measured by thin layer chromatography(TLC).We cloned a β-xylosidase gene, the open reading frame of which is1680bp, encoding559amino acids. The encoding β-xylosidase has a speculated molecular weight of63.2KDa and a isoelectric point of4.90. The purified recombinant enzyme showed optimal activity at pH6.2and50℃and was stable at pH5.0-8.0and45-50℃. The half-life temperature of the enzyme was50℃when the enzyme was incubated in different temperature for1h. The specific activity of the purified recombinant enzyme was618.5mU/mg, and the Km and Vmax was2.43mmol/L and20.17μmol/(min-mg). The recombinant enzyme was sensitive to xylose (inhibitor constant Ki=24.04mmol/L) and the enzyme was inhibited by PMSF, Co2Fe3+,Cu2+, Hg2+, Zn2+and SDS(100%), but2mmol/L Fe2+can significantly increase the enzyme activity(164%). The enzyme couldnot hydrolyze the7-xylose-10-deacetyl taxol to10-deacetyl taxol, therefor we suggested there is another xylosidase which can hydrolyze the7-xylose-10-deacetyl taxol to10-deacetyl taxol in Enterobacter sp. CGMCC2487.