Liang Yuanmei,Hou Juan,Liu Ying,Luo Yifan,Tang Jie,Cheng Jay J.,Daroch Maurycy
Cyanobacteria are prokaryotic phototrophs capable of achieving high cellular densities with minimal inputs. These prokaryotic organisms can grow using sunlight as energy source and carbon dioxide as carbon source what makes them promising candidates as microbial cell factories for the production of numerous compounds such as chemicals, fuels, or biocatalysts. In this study, we have successfully designed and constructed using synthetic biology approach two recombinant strains of Synechococcus elongatus PCC7942 for heterologous expression of the industrially relevant Bacillus subtilis CotA laccase. One of the strains (PCC7942-NSI-CotA) was constructed through integration of the laccase gene into neutral site I of the cyanobacterial genome whilst the other (PCC7942-NSII-CotA) targeted neutral site II of the genome. Of the two strains the one with CotA laccase integrated in neutral site II (PCC7942-NSII-CotA) was superior in terms of growth rate and enzymatic activity toward typical laccase substrates: ABTS [2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonate)] and syringaldazine. That may suggest that two of the traditionally used neutral sites of S. elongatus PCC7942 are not equally suitable for the expression of certain transgenes. The PCC7942-NSII-CotA produced protein was capable of decolourising three classes of dyes namely: anthraquinonic-, azo-, and indigoid-type over 7 days of incubation making the strain a potentially useful microbial cell factory for the production of broad-spectrum biodegradation agent. Interestingly, presence of additional synthetic redox mediator ABTS had no effect on the degradation of these dyes.
Multi-spectroscopic and molecular docking methods were used to study the interaction between triclosan (TCS) and bovine serum albumin (BSA). The results indicated that the fluorescence quenching of BSA by TCS was due to the formation of TCS–BSA complex through static quenching. This result was also demonstrated by time-resolved fluorescence experiment. The binding constants and number of binding sites between TCS and BSA were 1.30 × 105 M−1 and 1.17 at 298 K, respectively. The thermodynamic parameters were studied in detail which suggested that hydrophobic forces and hydrogen bond played major roles in the TCS–BSA interaction. Moreover, the site marker competitive experiments and docking studies revealed that TCS could bind BSA into site I in subdomain IIA. All the results of UV–vis spectrophotometry, circular dichroism spectroscopy and synchronous fluorescence spectroscopy showed that interaction between TCS and BSA induced conformation changes of BSA.
Junsong Qin,Zhepeng Chen,Chuangyan Wang,Lin Mai,Xian Wang,Junfeng Li,Hui Liu,Yun Song
This study systematically explored the interaction mechanisms between two KRAS G12C inhibitors (Sotorasib and Adagrasib) and human serum albumin (HSA) via UV-vis spectroscopy, fluorescence spectroscopy, three-dimensional fluorescence spectroscopy, and molecular docking methods. The experimental findings demonstrated that both drugs caused static quenching of HSA fluorescence, with binding constants of 13.64 × 103M−1(Sotorasib) and 63.67 × 103M−1(Adagrasib), demonstrating significant selectivity differences in their binding affinities. UV spectral analysis demonstrated distinct microenvironmental perturbations: Sotorasib and Adagrasib induced a shift (∆λ = 7 nm and ∆λ = 8 nm, respectively) at 211 nm, consistent with altered polarity in HSA’s binding pockets. Fluorescence spectroscopy confirmed a 1:1 binding stoichiometry, with Stern-Volmer analysis validating static quenching as the dominant mechanism. Three-dimensional fluorescence spectra further highlighted Adagrasib’s stronger conformational impact, reducing tyrosine and tryptophan residue fluorescence intensities by 16% (Peak 1) and 10% (Peak 2), respectively, compared to Sotorasib. Molecular docking revealed divergent binding modes: Sotorasib occupied Sudlow Site I via three hydrogen bonds and hydrophobic interactions (∆G = −24.60 kJ·mol−1), whereas Adagrasib bound through one hydrogen bond and hydrophobic forces (∆G = −30.92 kJ·mol−1), with stability differences attributed to structural characteristics. This study uses multispectral technology and molecular docking to reveal the binding mechanism of Sotorasib and Adagrasib with HSA, providing a theoretical basis for designing highly targeted albumin nanocarriers. The strong binding properties of Adagrasib and HSA may reduce the toxicity of free drugs, providing direction for the development of long-acting formulations.
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