Sonneoneal0615

Laccases or benzenediol oxygen oxidoreductases (EC 1.10.3.2) are polyphenol multicopper oxidases that are known for their structural and functional diversity in various life forms. In the present study, the molecular and physico-chemical properties (redox-potential and secondary structures) of fungal laccase isozymes (FLIs) isolated from a medicinal mushroom Ganoderma lucidum were analyzed and compared with those of the recombinant bacterial laccases (rLac) obtained from different Yersinia enterocolitica strains. It was revealed that the FLIs contained His-Cys-His as the most conserved residue in its domain I Cu site, while the fourth and fifth residues were variable (Ile, Leu, or Phe). Evidently, the cyclic voltammetric measurements of Glac L2 at Type 1 Cu site revealed greater E° for ABTS/ABTS+ (0.312 V) and ABTS+/ABTS2+ (0.773 V) compared to the E° of rLac. Furthermore, circular dichroism-based conformational analysis revealed structural stability of the FLIs at acidic pH (3.0) and low temperature (70 °C). The zymographic studies further confirmed the functional inactivation of FLIs at high temperatures (≥70 °C), predominantly due to domain unfolding. These findings provide novel insight into the evolution of the catalytic efficiency and redox properties of the FLIs, contributing to the existing knowledge regarding stress responses, metabolite production, and the biotechnological utilization of metabolites.Accumulating evidence indicates that plant cell wall-associated receptor-like kinases (WAKs) involve in defense against pathogen attack, but their related signaling processes and regulatory mechanism remain largely unknown. We identified a WAK-like kinase (GhWAKL) from upland cotton (Gossypium hirsutum) and characterized its functional mechanism. Expression of GhWAKL in cotton plants was induced by Verticillium dahliae infection and responded to the application of salicylic acid (SA). Knockdown of GhWAKL expression results in the reduction of SA content and suppresses the SA-mediated defense response, enhancing cotton plants susceptibility to V. dahliae. And, ecotopic overexpression of GhWAKL in Arabidopsis thaliana conferred plant resistance to the pathogen. Further analysis demonstrated that GhWAKL interacted with a cotton DnaJ protein (GhDNAJ1) on the cell membrane. Silencing GhDNAJ1 also enhanced cotton susceptibility to V. dahliae. Moreover, the mutation of GhWAKL at site Ser628 with the phosphorylation decreased the interaction with GhDNAJ1 and compromised the plant resistance to V. dahliae. We propose that GhWAKL is a potential molecular target for improving resistance to Verticillium wilt in cotton.Mesorhizobium loti carbonic anhydrase (MlCA), an intrinsically high catalytic enzyme, has been employed for carbon dioxide capture and sequestration. However, recombinant expression of MlCA in Escherichia coli often forms inclusion bodies. Hence, protein partners such as fusion-tags and molecular chaperones are involved in regarding reduce the harshness of protein folding. TrxA-tag and GroELS have been chosen to co-express with MlCA in E. coli under an inducible T7 promoter or a constitutive J23100 promoter to compare productivity and activity. The results possessed that coupling protein partners effectively increased soluble MlCA up to 2.9-folds under T7 promoter, thus enhancing the CA activity by 120% and achieving a 5.2-folds turnover rate. Besides, it has also shifted the optimum temperature from 40 °C to 50 °C, promoted stability in the broad pH range (4.5 to 9.5) and the presence of various metal ions. Based on the in vitro assay and isothermal titration calorimetry (ITC) analysis, GroELS enhancing CA activity was due to change the intrinsic thermodynamic properties of the enzyme from endothermic to exothermic reaction (i.e., ∆H = 89.8 to -121.8 kJ/mol). Therefore, the collaboration of TrxA-MlCA with GroELS successfully augmented CO2 biomineralization.Solid-state is the preferred choice for storage of protein therapeutics to improve stability and preserve the biological activity by decreasing the physical and chemical degradation associated with liquid protein formulations. Lyophilization or freeze-drying is an effective drying method to overcome the instability problems of proteins. However, the processing steps (freezing, primary drying and secondary drying) involved in the lyophilization process can expose the proteins to various stress and harsh conditions, leading to denaturation, aggregation often a loss in activity of protein therapeutics. Stabilizers such as sugars and surfactants are often added to protect the proteins against physical stress associated with lyophilization process and storage conditions. Another way to curtail the degradation of proteins due to process related stress is by modification of the lyophilization process. Slow freezing, high nucleation temperature, decreasing the extent of supercooling, and annealing can minimize the formation of the interface (ice-water) by producing large ice crystals with less surface area, thereby preserving the native structure and stability of the proteins. Hence, a thorough understanding of formulation composition, lyophilization process parameters and the choice of analytical methods to characterize and monitor the protein instability is crucial for development of stable therapeutic protein products. BMS-927711 in vitro This review provides an overview of various stress conditions that proteins might encounter during lyophilization process, mechanisms to improve the stability and analytical techniques to tackle the proteins instability during both freeze-drying and storage.Herein, the immobilization of α-amylase onto hydroxyapatite (HA) and hydroxyapatite-decorated ZrO2 (10%wt) (HA-ZrO2) nanocomposite were investigated. The immobilization yield was 69.7% and 84% respectively. The structural differences were characterized using X-Ray diffraction, attenuated total reflectance-Fourier transform infrared spectra, Raman, and scanning electron microscope. After 10 repeated cycles, the residual activity of immobilized α-amylase onto HA and HA-ZrO2 nanocomposite was 46% and 70%, respectively. The storage stability was recorded to be 27%, 50% and 69% from its initial activity in the case of free and immobilized enzyme onto HA and HA-ZrO2 nanocomposite, respectively after 8 weeks. The pH-activity profile and temperature revealed pH 6.0 and temperature 50 °C as the optimal values of free α-amylase, while the optimum values for α-amylase on HA and HA-ZrO2 was shifted to pH 6.5 and 60 °C after immobilization. The immobilized α-amylase onto HA-ZrO2 showed comparatively higher catalytic activity than the free α-amylase.