APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
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2019
·Vol. 104
·期 2
·765-774
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IF 3.67
Tang Dandan,Liu Wei,Huang Lei,Cheng Leming,Xu Zhinan
25-hydroxyvitamin D 3 has attracted considerable attention due to its great medical value and huge market demand in animal husbandry. Microbial production of 25-hydroxyvitamin D 3 has been recognized as an alternative superior to traditional chemical synthesis. In this study, a Gram-positive bacteria zju 4-2 (CCTCC M 2019385) was isolated from the soil using vitamin D 3 as the sole carbon source and was identified as Bacillus cereus according to its physiological characteristics and 16S rRNA analysis, which also showed a relatively high capacity for 25-hydroxyvitamin D 3 production. Through systematic optimization of different catalytic conditions, the optimal solvent system of vitamin D 3 , vitamin D 3 addition time and concentration, temperature, and pH were shown to be propylene glycol/ethanol (v/v = 9:1), early stationary phase, 2 g/L, 37 °C, and pH 7.2, respectively. With these optimal conditions, 796 mg/L of 25-hydroxyvitamin D 3 was achieved after 48 h bioconversion with zju 4-2 at the shake flask level. Finally, up to 830 mg/L 25-hydroxyvitamin D 3 with a yield of 41.5% was obtained in a 5 L fermentation tank. Our developed biotransformation process with this newly isolated strain provides a platform to produce 25-hydroxyvitamin D 3 efficiently at industrialization scale.
研究领域:
微生物学
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生物技术
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医药科学
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DOI
PubMed
高影响因子
ACS Synthetic Biology
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2025
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IF 3.9
Jiaying Ai,Ziyang Yin,Jikai Gao,Wenjing Wang,Fuping Lu,Hui-Min Qin,Shuhong Mao
P450 enzymes are promising biocatalysts and play an important role in the field of drug synthesis due to their high catalytic activity and stereoselectivity. CYP109E1 from Bacillus megaterium was used to convert VD3 for the production of 25(OH)VD3. However, the industrial production was still limited due to the low catalytic performance of CYP109E1. To overcome this, we constructed an engineered strain containing a modified CYP109E1 coupled with an efficient electron transfer chain and NADPH regeneration system. First, Adx4–108T69E-Fpr was identified as the most compatible redox partner for the enzyme based on in-silico analysis. Then, targeted mutations were introduced at the substrate channel of CYP109E1, resulting in higher production efficiency. Next, the production of 25(OH)VD3 was increased by 13.1% after introducing a double Adx4–108T69E expression cassette. Finally, an NADPH regeneration system was introduced by overexpressing zwf, which increased the yield of 25(OH)VD3 48.7%. These results demonstrate that recombinant Escherichia coli BL21 (DE3) coexpressing CYP109E1_R70A-ZWF and 2Adx4–108T69Es-Fpr is an efficient whole-cell biocatalyst for the synthesis of 25(OH)VD3, illustrating an attractive strategy for improving the catalytic efficiency of P450 enzymes.
研究领域:
生物化学
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药物合成
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酶工程
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产品关联
DOI
PubMed
高影响因子
Journal of Analysis and Testing
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2025
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IF 7
Chen Huaiyi,Liao Ziwei,Sun Gongwei,Wu Tianhao,Yang Jinlei,Pan Siyuan,Zhang Sichun,Tang Fei
Due to the important role of vitamin D in metabolism, developing a method for rapid and high-throughput analysis of vitamin D and its metabolites is crucial. However, the current clinical diagnostic methods have the disadvantage of complicated operating procedures. In the present study, a technique combining immunocapture with mass spectrometry for rapidly detecting of 25-hydroxyvitamin D 3 in serum was developed. This method employs an antibody–antigen reaction to specifically enrich 25-hydroxyvitamin D 3 and remove complex interfering substances. Qualitative and quantitative analysis of 25-hydroxyvitamin D 3 was achieved by nano-electrospray ionization-mass spectrometry (nano-ESI–MS). The method exhibited good linearity in the range of 1–100 ng/mL with satisfactory recovery and repeatability. The proposed method is promising for high-throughput and rapid analysis of small molecules with mass spectrometry. And it can potentially be combined with portable mass spectrometers to meet the requirements of point-of-care detection.
研究领域:
医学
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分析化学
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产品关联
DOI
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2025
·
IF 6.2
Ziqi Liang,Qiao Zhou,Yicheng Li,Xiaoqing Liu,Yiwen Shen,Jian Tian,Xiaolu Wang,Xing Qin,Yuan Wang,Huiying Luo,Xiaojun Yang,Bin Yao,Tao Tu
Cytochrome P450 enzymes (P450s) are promising candidates for the biosynthesis of 25-hydroxyvitamin D3 (25(OH)VD3). However, their industrial application is limited by challenges, such as low stability, inefficient catalysis, and uncoupling reactions. The construction of self-sufficient P450s offers a strategic solution to these limitations, but requires linker optimization to regulate interdomain conformational dynamics. In this study, we integrated whole-cell biocatalyst screening with systematic optimization of reaction conditions, including cosolvents, cell concentrations, and plasmid selection, to enhance catalytic performance. Under optimized conditions, the heme domain Vdh-K1 achieved a 91.6% conversion efficiency and was subsequently selected for chimeric enzyme assembly. By employing local energetic frustration analysis to evaluate protein flexibility and allosteric dynamics, we identified chimeric P450 variants with highly frustrated linkers. The optimal variant, VK1-CYP116B46-L21, exhibited improved thermostability, catalytic activity, and coupling efficiency, achieving a yield of 4.89 mM (1.96 g/L) 25(OH)VD3 in Escherichia coli whole-cell catalysis─the highest reported yield to date. This work underscores the utility of computational frustration analysis in refining linker dynamics for multidomain enzymes and establishes a scalable, cost-effective framework to advance P450s systems for industrial biosynthesis of high-value compounds.
研究领域:
生物化学
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工业生物技术
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酶工程
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF SEPARATION SCIENCE
·
2025
·Vol. 48
·期 4
·e70130
·
IF 2.8
Qing Han,Min Zhou,Xuejun Kang,Guozhe Deng,Feng Guo,Li Geng
Vitamin D plays a crucial role in skeletal metabolism and is implicated in various diseases. Several studies have indicated that human vitamin D status can be evaluated through urine hydroxyvitamin D levels, in addition to plasma hydroxyvitamin D concentrations. In this study, we developed a novel method for the detection of trace amounts of 25-hydroxyvitamin D 3 [25(OH)D 3 ] and 24,25-dihydroxyvitamin D 3 [24,25(OH) 2 D 3 ] in urine. This method is based on high-performance liquid chromatography coupled with atmospheric pressure chemical ion source-tandem mass spectrometry, integrated with packed-fibers solid-phase extraction. The composite polystyrene/graphene oxide (PS/GO) nanofibers were synthesized as the sorbent for concentrating 25(OH)D 3 and 24,25(OH) 2 D 3 , achieving a twentyfold increase in sensitivity when analyzing human urine samples. Fourier-transform infrared spectroscopy, X-ray diffraction, and energy-dispersive X-ray spectroscopy mapping analysis were employed to characterize GO, PS, and PS/GO. The results indicated that the composite nanofibers were successfully synthesized. In addition, we investigated the relationship between the extraction performance of the fibers and the electrospinning process at varying flow rates of spinning solution through morphological studies and Brunauer-Emmett-Teller surface area analysis. During the extraction process, purification, concentration, and desorption were accomplished within a single step. The established method demonstrated excellent sensitivity and efficiency. Under optimal conditions, limits of detection (signal-to-noise ratio = 3) were found to be 0.33 ng/mL for 25(OH)D 3 and 0.19 ng/mL for 24,25(OH) 2 D 3 ; furthermore, linearity was deemed acceptable across urine samples. Recovery rates ranged from 89.5% to 109.7% for 25(OH)D 3 and from 90.3% to 103.1% for 24,25(OH) 2 D 3 , respectively.
研究领域:
分析化学
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医学检验
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营养学
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL SCIENCES
·
2024
·
IF 1.8
Cai Tongji,Chen Meilun,Yang Jie,Tang Chunhua,Lu Xiaoling,Wei Zheng,Jiang Hanbing,Hou Yucui,Zhao Jia,Yu Peng
Abstract Vitamin D 3 (VD 3 ) is the main form of vitamin D and an essential nutrient for maintaining human life. Currently, traditional methods for detecting 25-hydroxyvitamin D3(25(OH)D 3 ) are complex and expensive. In this study, we constructed an accurate, sensitive, simple, and cost-effective label-free biosensor based on an aptamer for the detection of 25(OH)D 3 . The aptamer-modified sulfhydryl adopted self-assembly as a way to stably immobilize at the glassy carbon electrode (GCE) surface modified by gold nanoparticles (AuNPs). Upon 25(OH)D 3 binding to the aptamer, the complexes inhibit electron transfer at the electrode surface, leading to reduced [Fe(CN) 6 ] 3−/4− redox peak current. Consequently, the quantity of 25(OH)D 3 that interacts with the electrode-bound aptamer correlates with the observed electric current response values. The Aptamer/AuNPs/GCE aptasensor achieved direct and highly sensitive detection of 25(OH)D 3 over a wide concentration range (1.0–1000 nM), with a limit of detection of 1.0 nM. At the same time, other molecules with a similar structure, such as 25(OH)D 2 , Vitamin D 3 , and Vitamin D 2 , had lower response interference than 25(OH)D 3 . Therefore, this biosensor has great potential to become a portable diagnostic device for 25(OH)D 3 . Graphic abstract
研究领域:
分析化学
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生物传感器
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纳米技术
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产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）