Expression and characterization of a novel cytochrome P450 enzyme from Variovorax paradoxus S110 / 生物工程学报

Cytochrome P450 monooxygenases as powerful biocatalysts catalyze a wide range of chemical reactions to facilitate exogenous substances metabolism and biosynthesis of natural products. In order to explore new catalytic reactions and increase the number of P450 biocatalysts used in synthetic biology, a new self-sufficient cytochrome P450 monooxygenase (P450(VpMO)), belongs to CYP116B class, was mined from Variovorax paradoxus S110 genome and expressed in Escherichia coli. Based on characterization of the enzymatic properties, it shows that the optimal pH and temperature for P450(VpMO) reaction activity are 8.0 and 45 °C, respectively. P450(VpMO) is relatively stable at temperatures below 35 °C. The Km and kcat of P450(VpMO) toward 4-Methoxyacetophenone are 0.458 mmol/L and 2.438 min⁻¹, respectively. Importantly, P450(VpMO) was able to catalyze the demethylation reaction for a range of substrates containing methoxy group. Its demethylation reactivity is reasonably better than other P450s belongs to CYP116B class, particularly, for 4-methoxyacetophenone with a great conversion efficiency at 91%, showing that P450(VpMO) could be used as a great biocatalyst candidate for further analysis.

Derivative Spectrophotometric Methods for the Determination of Bendamustine Hydrochloride.

Bendamustine hydrochloride is used to treat chronic lymphocytic leukemia. It kills the existing cancer cells and limits the growth of new cancer cells. Three simple, rapid and sensitive spectrophotometric methods were developed for the determination of Bendamustine hydrochloride in phosphate buffer (pH 8.0) (Method A) and boric buffer (pH 9.0) (Method B). Method C is a difference spectroscopy technique in which the amplitude was chosen for the analytical calculations. Bendamustine hydrochloride obeys Beer-Lambert’s law over the concentration range 1-40 μg/ml, 0.1-40 μg/ml and 5- 40 μg/ml with regression equations y = 0.003x + 0.001(r² = 0.998) y = 0.0027x + 0.0005 (r² = 0.999), and y = 0.0034x + 0.006 (r² = 0.994) for Method A, B and C respectively. The methods were validated as per ICH guidelines and can be applied for the determination of Bendamustine hydrochloride in pharmaceutical formulations.

Spectroscopic studies on the interaction of bilirubin with symmetrical alkyl diamines.

The interactions of symmetrical alkyldiamines with bilirubin-IX α have been examined in dichloromethane and dioxane solutions, by visible region difference spectroscopy and florescence methods. In dioxane solutions a clear difference is observed between the complexes of the shorter chain diamines (number of spacer methylene groups (n ≤ 4)) and the longer chain diamines (n ≥ 6). The variations in spectral features with diamine chain length are less pronounced in dichloromethane. The spectroscopic results are consistent with the occurrence of distinct bilirubin conformations depending upon the solvent and the geometry of the interacting receptor. Based on molecular modelling two conformations are proposed. A ‘ridge-tile’ model similar to that observed in crystals is favoured for binding to the longer diamines, while a ‘quasi-cyclic’ structure is preferred for interaction with the short chain diamines.