Development of a Universal One-Step Purification and Activation Method to Engineer Protein-Glutaminase through Rational Design.

Cytotoxic enzymes often exist as zymogens containing prodomains to keep them in an inactive state. Protein-glutaminase (PG), which can enhance various functional characteristics of food proteins, is an enzyme containing pro-PG and mature-PG (mPG). However, poor activity and stability limit its application while tedious purification and activation steps limit its high-throughput engineering. Here, based on structural analysis, we replaced the linker sequence between pro-PG and mPG with the HRV3C protease recognition sequence and then coexpressed it with HRV3C protease in Escherichia coli to develop an efficient one-step purification and activation method for PG. We then used this method to obtain several mutants designed by a combination of computer-aided approach and beneficial point mutations. The specific activity (131.6 U/mg) of the best variant D1 was 4.14-fold that of the wild type, and t1/2 and T5010 increased by 13 min and 7 °C, respectively. D1 could effectively improve the solubility and emulsification of wheat proteins, more than twice the effect of the wild type. We also discussed the mechanism underlying the improved properties of D1. In summary, we not only provide a universal one-step purification and activation method to facilitate zymogen engineering but also obtain an excellent PG mutant.

A Heterogeneous Single Atom Cobalt Catalyst for Highly Efficient Acceptorless Dehydrogenative Coupling Reactions.

A fundamental understanding of metal active sites in single-atom catalysts (SACs) is important and challenging in the development of high-performance catalyst systems. Here, a highly efficient and straightforward molten-salt-assisted approach is reported to create atomically dispersed cobalt atoms supported over vanadium pentoxide layered material, with each cobalt atom coordinated with four neighboring oxygen atoms. The liquid environment and the strong polarizing force of the molten salt at high temperatures potentially favor the weakening of VO bonding and the formation of CoO bonding on the vanadium oxide surface. This cobalt SAC achieves extraordinary catalytic efficiency in acceptorless dehydrogenative coupling of alcohols with amines to give imines, with more than 99% selectivity under almost 100% conversion within 3 h, along with a high turnover frequency (TOF) of 5882 h-1 , exceeding those of previously reported benchmarking catalysts. Moreover, it delivers excellent recyclability, reaction scalability, and substrate tolerance. Density functional theory (DFT) calculations further confirm that the optimized coordination environment and strong electronic metal-support interaction contribute significantly to the activation of reactants. The findings provide a feasible route to construct SACs at the atomic level for use in organic transformations.

Novel biosynthesis, characterization and bio-catalytic potential of green algae (Spirogyra hyalina) mediated silver nanomaterials.

In recent years green nanotechnology gained significant importance to synthesize nanoparticles due to their cost effectiveness and biosafety. In the current study, silver nanoparticles were synthesized by using extract of Spirogyra hyalina as a capping and reducing agent. The synthesized nanoparticles were characterized by UV-Visible spectroscopy, Fourier transform infrared spectroscopy, Scanning electron microscopy, energy dispersive X-ray spectroscopy, and X-ray diffractive analysis. Silver nanoparticles give a characteristic Surface Plasmon Resonance peak of 451 nm at 2.21 a.u (arbitrary unit). SEM micrograph revealed the spherical morphology and average grain size of 52.7 nm. Furthermore, antibacterial, antifungal, insecticidal, antioxidant and membrane damage activities were determined. The maximum antibacterial and antifungal activity was observed for Pseudomonas aeruginosa (18 ± 1.2 mm) and Fusarium solani (14.3 ± 0.6 mm), respectively. In membrane damage assay, Pseudomonas aeruginosa absorbed A260 wavelength and gave maximum peak values of 0.286, 0.434 and 0.629 at 25, 35 and 45 µg/mL of silver nanoparticles. The membrane damage assay confirmed that nanoparticles are involved in bacterial cell membrane damage. At 500 ppm silver nanoparticles showed 30% mortality against Tribolium castaneum (a common grain pest). The silver nanoparticles also showed potent antioxidant activity and successfully scavenged the DPPH free radicals upto 53.43 ± 0.17, 43.26 ± 0.97, 31.39 ± 0.33, 24.62 ± 0.85, and 14.13 ± 0.12% at a concentration of 400, 200, 100, 50, and 25 µg/mL of nanoparticles, respectively. It is concluded that silver nanoparticles can easily be synthesized by using green algae Spirogyra hyalina as a capping and reducing agent. Silver nanoparticles showed potent biomedical activities and thus can be used for therapeutic applications invitro and invivo.