Lixin Kang,Haoran Li,Kexin Lin,Shen Hu,Song Liu,Yuben Qiao,Ying Wang,Aitao Li
The biocatalytic production of 25-hydroxyvitamin D3 (25(OH)VD3, calcidiol) represents a superior alternative to traditional chemical synthesis. However, the reported VD3 hydroxylases generally exhibit suboptimal catalytic efficiency, limiting their practical applications. In this study, a cytochrome P450 CYP107Pdh from Pseudonocardia dioxanivorans_CB1190, which displays unique C26 hydroxylation activity on VD3, was identified. Then, structure-guided loop engineering combined with binding pocket reshaping was conducted on CYP107Pdh, leading to the generation of the quintuple variant 89_90insIP/T112A/V161L/G186V (M4). This variant shifted the regioselectivity from 91% C26 in the wild type (WT) to 81% C25 for calcidiol production. In addition, variant M4 showed a remarkable enhancement in catalytic activity, achieving a catalytic efficiency (kcat/Km) that is 75-fold higher than that of the WT. Computational analyses revealed that the regioselectivity shift and activity improvement are primarily attributed to a conformational transition in the substrate-binding pocket from an open to a more closed state, which optimizes substrate binding and facilitates efficient 25-hydroxylation of VD3. Finally, a semipreparative biotransformation yielded 2.64 g of crystalline calcidiol (95% purity) from a 1-L reaction, thereby expanding the enzyme library of VD3 hydroxylases and underscoring its potential for industrial-scale production of calcidiol.
Ning Gao,Yuhan Fu,Hexin Gong,Haotian Liu,Weinan Li
Recently, growing and conclusive evidences showed the particularly close ties between the vitamin D (VD) and breast cancer (BC). However, few researches were focused on the delivery system against breast cancer which based on cholecalciferol (VD 3 ). Herein, we designed a series of self-assembled micelles. The conjugate (HA-VD) of VD 3 with hyaluronic acid (HA) actively targeting breast cancer can efficiently delivery doxorubicin (DOX). The HA-VD conjugate was successfully synthesized, which was confirmed using 1 H nuclear magnetic resonance (NMR) and Fourier transform infrared (FITR). Preliminary studies of its physical and chemical properties indicated that VD 3 was successfully grafted with the HA (HAVD). When the molar ratio was 1:2 and the degree of substitution (DS) was 18.6%, the minimum critical micelle concentration (CMC) value was 0.0137 mg/mL. For DOX-loaded HAVD micelles (DOX/HAVD), the drug loading (DL) was 6.2% and drug encapsulation efficiency (EE) was 79.5%. The DOX/HAVD micelles remain stable in serum for 48 h. The in vitro release rate of DOX decreased after encapsulating compared with that without HAVD encapsulation. The decrease with IC 50 of DOX/HAVD micelle was significantly stronger than that of free DOX ( p < 0.05) and blank micelles ( p < 0.01). At 50% cell inhibition rate, VD 3 and DOX acted synergistically on BC cells. Hence, the HAVD micelles can significantly improve the therapeutic effect of DOX against BC cells. In a word, the DOX/HAVD micelles have great potential in treating breast cancer.
危险品化学品经营许可证（带存储）