Characterization of a novel carboxylesterase from Streptomyces lividans TK24 and site-directed mutagenesis for its thermostability.

As an industrial enzyme that catalyzes the formation and cleavage of ester bonds, carboxylesterase has attracted attention in fine chemistry, pharmaceutical, biological energy and bioremediation fields. However, the weak thermostability limits their further developments in industrial applications. In this work, a novel carboxylesterase (EstF) from Streptomyces lividans TK24, belonging to family XVII, was acquired by successfully heterologous expressed and biochemically identified. The EstF exhibited optimal activity at 55 °C, pH 9.0 and excellent catalytic performances (Km = 0.263 mM, kcat/Km = 562.3 s-1 mM-1 for p-nitrophenyl acetate (pNPA2) hydrolysis). Besides, the EstF presented exceptionally high thermostability with a half-life of 387.23 h at 55 °C and 2.86 h at 100 °C. Furthermore, the EstF was modified to obtain EstFP144G using the site-directed mutation technique to investigate the effect of single glycine on thermostability. Remarkably, the mutant EstFP144G displayed a 5.10-fold increase of half-life at 100 °C versus wild-type without affecting catalytic performance. Structural analysis implied that the glycine introduction could release a steric strain and induce cooperative effects between distal residues to increase the thermostability. Therefore, the thermostable EstF and EstFP144G with prominently catalytic characteristics have potential industrial applications and the introduction of a single glycine strategy opens up alternative avenues for the thermostability engineering of other enzymes.

NAD (P)H Quinone Dehydrogenase 1-Targeting Triptolide Analogue Causes Tumor Regression and Sensitizes Cisplatin-Resistant Lung Cancer to Chemotherapy.

Cisplatin (DDP) is a first-line chemotherapeutic drug against lung cancer. Nonetheless, the effectiveness of this drug is hampered by drug resistance. Overcoming drug resistance is crucial for improving the outcomes of lung cancer treatment. Here, we reported the effect of CX-23, an activated triptolide analogue that targets NAD (P)H quinone dehydrogenase 1 (NQO1), on DDP-resistant lung cancer and sensitizes DDP-resistant lung cancer to chemotherapy. Our findings unveiled the antiproliferative activity of CX-23 against both A549- and DDP-resistant A549 (A549/DDP) cells while enhancing the chemosensitivity of these cells to DDP. Notably, CX-23 demonstrated no toxicity toward normal lung cells. Further investigations revealed that CX-23 exerts its effects by blocking AKT phosphorylation, leading to reduced expression of Mcl-1 and Bcl-2, and upregulating cleaved-caspase-3 levels, ultimately inducing apoptosis in cancer cells. CX-23 can be rapidly transformed in both A549 and A549/DDP cell lysates while remaining stable in mouse plasma and normal lung tissues. Pharmacokinetic analysis showed that CX-23 can distribute to lung tissues. Moreover, in vivo studies showed that CX-23 can prevent DDP-resistant lung cancer progression without causing any toxicity in the liver, kidneys, or lungs after 6 weeks of treatment. The combination of CX-23 and DDP not only significantly inhibited tumor progression compared to DDP alone but also attenuated DDP-induced kidney toxicity. These findings suggest that CX-23 alone or in combination with DDP may provide an alternative therapeutic option for DDP-resistant lung cancer.

Dual-functional cellulase-mediated gold nanoclusters for ascorbic acid detection and fluorescence bacterial imaging.

Protein-protected metal nanomaterials are becoming the most promising fluorescent nanomaterials for biosensing, bioimaging, and therapeutic applications due to their obvious fluorescent molecular properties, favorable biocompatibility and excellent physicochemical properties. Herein, we pioneeringly prepared a cellulase protected fluorescent gold nanoclusters (Cel-Au NCs) exhibiting red fluorescence under the excitation wavelength of 560 nm via a facile and green one-step method. Based on the fluorescence turn-off mechanism, the Cel-Au NCs were used as a biosensor for specificity determination of ascorbic acid (AA) at the emission of 680 nm, which exhibited satisfactory linearity over the range of 10-400 µM and the detection limit of 2.5 µM. Further, the actual sample application of the Au NCs was successfully established by evaluating AA in serum with good recoveries of 98.76%-104.83%. Additionally, the bacteria, including gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) and gram-negative bacteria (Escherichia coli), were obviously stained by Cel-Au NCs with strong red emission. Thereby, as dual-functional nanoclusters, the prepared Cel-Au NCs have been proven to be an excellent fluorescent bioprobe for the detection of AA and bacterial labeling in medical diagnosis and human health maintenance.

Reusable carboxylesterase immobilized in ZIF for efficient degradation of chlorpyrifos in enviromental water.

The past few decades have witnessed biodegradation of pesticides as a significant method in remediation of the environment for its specificity, efficiency and biocompatibility. However, the tolerability and recyclability of the enzymes in pesticide degradation and the development of enzymes that biodegrad pesticides are still urgent problems to be solved so far. Herein, a novel hyper-thermostable and chlorpyrifos-hydrolyzing carboxylesterase EstC was immobilized by biomineralization using zeolitic imidazolate framework (ZIF), one of the metal-organic frameworks (MOFs) with highly diverse structure and porosity. Compared with free enzyme, EstC@ZIF with a cruciate flower-like morphology presented scarcely variation in catalytic efficiency and generally improved the tolerance to organic solvents or detergents. Furthermore, there was scarcely decrease in the catalytic efficiency of EstC@ZIF and it also showed good reusability with about 50% residual activity after 12 continuous uses. Notably, EstC@ZIF could be used in actual water environment with an excellent value of degradation rate of 90.27% in 120 min, and the degradation efficiency remained about 50% after 9 repetitions. The present strategy of immobilizing carboxylesterase to treat pesticide-contaminated water broadens the method of immobilized enzymes on MOFs, and envisions its recyclable applicability in globe environmental remediation.