LXYL-P1-2 immobilized on magnetic nanoparticles and its potential application in paclitaxel production

BACKGROUND: LXYL-P1-2 is the first reported glycoside hydrolase that can catalyze the transformation of 7-b-xylosyl-10-deacetyltaxol (XDT) to 10-deacetyltaxol (DT) by removing the D-xylosyl group at the C7 position. Successful synthesis of paclitaxel by one-pot method combining the LXYL-P1-2 and 10- deacetylbaccatin III-10-b-O-acetyltransferase (DBAT) using XDT as a precursor, making LXYL-P1-2 a highly promising enzyme for the industrial production of paclitaxel. The aim of this study was to investigate the catalytic potential of LXYL-P1-2 stabilized on magnetic nanoparticles, the surface of which was modified by Ni2+-immobilized cross-linked Fe3O4@Histidine. RESULTS: The diameter of matrix was 20­40 nm. The Km value of the immobilized LXYL-P1-2 catalyzing XDT (0.145 mM) was lower than that of the free enzyme (0.452 mM), and the kcat/Km value of immobilized enzyme (12.952 mM s 1 ) was higher than the free form (8.622 mM s 1 ). The immobilized form maintained 50% of its original activity after 15 cycles of reuse. In addition, the stability of immobilized LXYL-P1-2, maintained 84.67% of its initial activity, improved in comparison with free form after 30 d storage at 4 C. CONCLUSIONS: This investigation not only provides an effective procedure for biocatalytic production of DT, but also gives an insight into the application of magnetic material immobilization technology.

Study on methods for screening anti-tumor active components of Taxus chinensis based on integrity of traditional Chinese medicine / 中草药

Objective: Two screening methods for drug active constituents were established based on the integrity of traditional Chinese medicine to screen the anti-breast cancer active components, which were hollow fiber cell fishing-high performance liquid chromatography (HFCF-HPLC) and cell fishing-dispersive liquid-liquid microextraction-high performance liquid chromatography (CF-DLLME-HPLC). Methods: According to HFCF-HPLC, MCF-7 cells were planted into the cavity of polypropylene hollow fiber, and then the hollow fiber was put into the extract of Taxus chinensis to screen and capture the bioactive component groups combined with cells in a simulated human body environment. According to CF-DLLME-HPLC, on the basis of normal cell metabolism, MCF-7 cells were co-incubated with T. chinensis extract, supernatant of different incubation time was taken for liquid phase microextraction, and then chromatographic analysis was performed to find potential active component groups according to chromatographic peak changes. Results: Anti-breast cancer active components of T. chinensis were obtained by these two methods. 10-Deacetylbaccatin III, baccatin III, taxinine M, 10-deacetyltaxol, cephalomannine, taxol, taxuyunnanine C were obtained by HFCF-HPLC, and 10-deacetyltaxol, baccatin III, taxinine M, 10-deacetyltaxol, sciadopitysin were obtained by CF-DLLME-HPLC. Conclusion: The two methods can screen the effective antitumor active ingredients, and provide a convenient, universal and efficient new method for the screening of active ingredients and the comprehensive evaluation of the quality of traditional Chinese medicine.