The Discovery, Molecular Cloning, and Characterization of Dextransucrase LmDexA and Its Active Truncated Mutant from Leuconostoc mesenteroides NN710.

Dextransucrases play a crucial role in the production of dextran from economical sucrose; therefore, there is a pressing demand to explore novel dextransucrases with better performance. This study characterized a dextransucrase enzyme, LmDexA, which was identified from the Leuconostoc mesenteroides NN710. This bacterium was isolated from the soil of growing dragon fruit in Guangxi province, China. We successfully constructed six different N-terminal truncated variants through sequential analysis. Additionally, a truncated variant, ΔN190LmDexA, was constructed by removing the 190 amino acids fragment from the N-terminal. This truncated variant was then successfully expressed heterologously in Escherichia coli and purified. The purified ΔN190LmDexA demonstrated optimal hydrolysis activity at a pH of 5.6 and a temperature of 30 °C. Its maximum specific activity was measured to be 126.13 U/mg, with a Km of 13.7 mM. Results demonstrated a significant improvement in the heterologous expression level and total enzyme activity of ΔN190LmDexA. ΔN190LmDexA exhibited both hydrolytic and transsaccharolytic enzymatic activities. When sucrose was used as the substrate, it primarily produced high-molecular-weight dextran (>400 kDa). However, upon the addition of maltose as a receptor, it resulted in the production of a significant amount of oligosaccharides. Our results can provide valuable information for enhancing the characteristics of recombinant dextransucrase and potentially converting sucrose into high-value-added dextran and oligosaccharides.

Magnetic CuFe2O4 with intrinsic protease-like activity inhibited cancer cell proliferation and migration through mediating intracellular proteins.

Protease has been widely used in biological and industrial fields. Developing efficient artificial enzyme mimics remains a major technical challenge due to the high stability of peptide bonds. Nanoenzymes with high stability, high activity and low cost, provided new opportunities to break through natural enzyme inherent limitations. However, compared with many nanomaterials with inherent peroxidase activity, the intrinsic mimic proteases properties of magnetic nanomaterials were seldom explored, let alone the interaction between magnetic nanomaterials and cellular proteins. Herein, we reported for the first time that magnetic CuFe2O4 possesses inherent protease activity to hydrolyze bovine serum albumin (BSA) and casein under physiological conditions, and the CuFe2O4 is more resistant to high temperature than the natural trypsin. It also exhibited significantly higher catalytic efficiency than other copper nanomaterials and can be recycled for many times. Protease participated in pathophysiological processes and all stages of tumor progression. Interesting, CuFe2O4 exhibited anti-proliferative effect on A549, SKOV3, HT-29, BABL-3T3 and HUVEC cells, as well as it was particularly sensitive against SKOV3 cells. CuFe2O4 was about 30 times more effective than conventional chemotherapy drugs oxaliplatin and artesunate against SKOV3 cells. In addition, CuFe2O4 also mediated the expression of intracellular proteins, such as MMP-2, MMP-9, F-actin, and NF-kB, which may be associated with global protein hydrolysis by CuFe2O4, leading to inhibition of cell migration. The merits of the high magnetic properties, good protease-mimic and antitumor activities make CuFe2O4 nanoparticles very prospective candidates for many applications such as proteomics and biotechnology.