Developed a signal-amplified fluorescence sensing method for rapid chymotrypsin activity monitoring. • Compared with other methods, this approach exhibits good sensitivity and selectivity. • Enhanced fluorescence sensing was combined with target fishing to construct a chymotrypsin inhibitor screening system. • This integrated platform quantifies chymotrypsin activity and identifies bioactive inhibitors from TCM extracts. Regulation and screening of biomacromolecule (e.g., enzyme) activity is one of the core tasks in disease treatment and drug discovery. Natural products are an important source of enzyme inhibitors, but their complex composition makes it difficult to develop effective candidate drug molecules. As a typical biomacromolecular target, chymotrypsin (CHT) exhibits aberrant activity closely associated with diseases such as wound healing, chronic pancreatitis, and emphysema. Therefore, efficient screening of enzyme inhibitors from natural products holds significant importance for clinical diagnosis, treatment, and drug development. This study establishes an integrated screening platform for enzyme inhibitors by combining a Cu 2+ -casein-mediated fluorescence-enhanced biosensing system with targeted ligand capture technology, enabling highly sensitive screening and precise identification of chymotrypsin inhibitors from natural products. In this platform, Cu 2+ -casein functions as a biomacromolecular substrate while also acting as a peroxidase-mimetic enzyme, significantly enhancing the fluorescence signal amplification effect of o -phenylenediamine oxidation. When chymotrypsin hydrolyzes casein, the structure of casein is disrupted, reducing its copper ion-binding capacity. Which leads to decreased catalytic activity and subsequent changes in fluorescence intensity, allowing quantitative detection of chymotrypsin activity with a detection limit as low as 13.6 ng mL −1 . In the targeted fishing module, immobilized chymotrypsin (CHT@NH 2 -SiO 2 ) is employed as an affinity medium, and combined with HPLC and LC-MS analysis, enables precise identification of active components in complex samples. This integrated strategy mitigates certain limitations of individual techniques, providing a feasible approach for efficient, high-throughput screening of inhibitors targeting biomacromolecules from complex systems. Download: Download high-res image (207KB) Download: Download full-size image
Zhongliang Sun,Shuonan Cao,Mengru Xin,Shoukai Guo,Yu Zhang,Qiang Wang,Liqin Sun
Microalgae are efficient biological platforms for CO 2 capture and renewable bioresource production; however, carbon assimilation efficiency remains a major bottleneck. In this study, symbiotic bacteria capable of secreting extracellular carbonic anhydrase (CA) were isolated from natural algal-associated environments to enhance CO 2 hydration and utilization in Chlorella sorokiniana . Among six CA-secreting isolates, three strains exhibited high extracellular CA activities and promoted algal growth without inhibitory effects. Co-cultivation with Bacillus safensis YD-1 and B. licheniformis YD-6 increased biomass productivity by up to 74 % and lipid content by 65–70 % compared with axenic controls, while mitigating total inorganic carbon (TIC) depletion during late-stage cultivation. The co-culture systems maintained extracellular CA activities of 3.6–4.8 U/mL and reduced TIC loss to 8.9–16.0 %, compared with 20.3–30.5 % in controls. Immobilization of YD-1 cells using alginate–calcium beads enabled repeated reuse over three cultivation cycles, retaining > 80 % of CA activity and sustaining consistent growth enhancement. These results demonstrate that immobilized CA-secreting symbiotic bacteria provide a self-regenerating and reusable biological strategy to alleviate carbon limitation, offering a practical and scalable approach for improving CO 2 utilization and biomass production in microalgal cultivation systems.
Meng-Meng Du,Yuan-Cheng Wang,Bao-Chang Sun,Yong Luo,Liang-Liang Zhang,Guang-Wen Chu,Hai-Kui Zou
Carbonic anhydrase (CA) is a high-efficiency biocatalyst that significantly improves the absorption of CO 2 by tertiary amine. This work aims to investigate kinetics behaviors from the perspective of enzymatic reaction mechanism. The influences of the CA concentration, type of tertiary amines, pH, and temperature on the reaction rate between CO 2 and tertiary amine ( ν ) and catalytic activity of CA ( φ ) were first investigated in a stopped-flow device. Adding 50 g∙m⁻³ CA enhanced ν in tertiary amine solutions by a factor ranging from 22 to 42 at 298 K and pH=9.5, demonstrating its excellent catalytic performance. The ν increased with increasing CA concentration, pH, temperature, and tertiary amine's pK a. φ increased with the increase of CA concentration, as well as the decrease of temperature, pH, and tertiary amine's pK a. Proteomics analysis further revealed that conformational changes of the CA's secondary structure induced by high pH and temperature altered the expressions of the local active-site region and deactivated CA, ultimately leading to a decrease in φ . Additionally, the CA-catalysis kinetics equation accorded with the Michaelis-Menten model, with catalytic second-order rate constants on the magnitude of 10 7 . Overall, this work provides a guideline for its industrial application in the CO 2 capture process.
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