Roles of the N-terminal motif in improving the activity and soluble expression of phenylalanine ammonia lyases in Escherichia coli.

Phenylalanine ammonia-lyase (PAL) has various applications in fine chemical manufacturing and the pharmaceutical industry. In particular, PAL derived from Anabaena variabilis (AvPAL) is used as a therapeutic agent to the treat phenylketonuria in clinical settings. In this study, we aligned the amino acid sequences of AvPAL and PAL derived from Nostoc punctiforme (NpPAL) to obtain several mutants with enhanced activity, expression yield, and thermal stability via amino acid substitution and saturation mutagenesis at the N-terminal position. Enzyme kinetic experiments revealed that the kcat values of NpPAL-N2K, NpPAL-I3T, and NpPAL-T4L mutants were increased to 3.2-, 2.8-, and 3.3-fold that of the wild-type, respectively. Saturation mutagenesis of the fourth amino acid in AvPAL revealed that the kcat values of AvPAL-L4N, AvPAL-L4P, AvPAL-L4Q and AvPAL-L4S increased to 4.0-, 3.7-, 3.6-, and 3.2-fold, respectively. Additionally, the soluble protein yield of AvPAL-L4K increased to approximately 14 mg/L, which is approximately 3.5-fold that of AvPAL. Molecular dynamics studies further revealed that maintaining the attacking state of the reaction and N-terminal structure increased the rate of catalytic reaction and improved the solubility of proteins. These findings provide new insights for the rational design of PAL in the future.

Computational design of α-amylase from Bacillus licheniformis to increase its activity and stability at high temperatures.

The thermostable α-amylase derived from Bacillus licheniformis (BLA) has multiple advantages, including enhancing the mass transfer rate and by reducing microbial contamination in starch hydrolysis. Nonetheless, the application of BLA is constrained by the accessibility and stability of enzymes capable of achieving high conversion rates at elevated temperatures. Moreover, the thermotolerance of BLA requires further enhancement. Here, we developed a computational strategy for constructing small and smart mutant libraries to identify variants with enhanced thermostability. Initially, molecular dynamics (MD) simulations were employed to identify the regions with high flexibility. Subsequently, FoldX, a computational design predictor, was used to design mutants by rigidifying highly flexible residues, whereas the simultaneous decrease in folding free energy assisted in improving thermostability. Through the utilization of MD and FoldX, residues K251, T277, N278, K319, and E336, situated at a distance of 5 Å from the catalytic triad, were chosen for mutation. Seventeen mutants were identified and characterized by evaluating enzymatic characteristics and kinetic parameters. The catalytic efficiency of the E271L/N278K mutant reached 184.1 g L-1 s-1, which is 1.88-fold larger than the corresponding value determined for the WT. Furthermore, the most thermostable mutant, E336S, exhibited a 1.43-fold improvement in half-life at 95 â„ƒ, compared with that of the WT. This study, by combining computational simulation with experimental verification, establishes that potential sites can be computationally predicted to increase the activity and stability of BLA and thus provide a possible strategy by which to guide protein design.

Combined metabolomics and gut microbiome to investigate the effects and mechanisms of Yuquan Pill on type 2 diabetes in rats.

Yuquan Pill (YQP) is a traditional Chinese medicine (TCM) for the treatment of type 2 diabetes (T2DM) in China for many years, and has a beneficial clinical effect. In this study, the antidiabetic mechanism of YQP was investigated for the first time from the perspective of metabolomics and intestinal microbiota. After 28 days of high-fat feeding, rats were injected intraperitoneally with streptozotocin (STZ, 35 mg/kg) followed by a single oral administration of YQP 2.16 g/kg and metformin 200 mg/kg for 5 weeks. The results showed that YQP was effectively improved insulin resistance and alleviated hyperglycemia and hyperlipidemia associated with T2DM. YQP was found to regulate metabolism and gut microbiota in T2DM rats using untargeted metabolomics and gut microbiota integration. Forty-one metabolites and five metabolic pathways were identified, including Ascorbate and aldarate metabolism, Nicotinate and nicotinamide metabolism, Galactose metabolism, Pentose phosphate pathway and Tyrosine metabolism. YQP can regulate T2DM-induced dysbacteriosis by modulating the abundance of Firmicutes, Bacteroidetes, Ruminococcus, Lactobacillus. The restorative effects of YQP in rats with T2DM have been confirmed and provide a scientific basis for the clinical treatment of diabetic patients.

Ultra-performance liquid chromatography-tandem mass spectrometry determination and pharmacokinetic studies of five phenolic acids in rat plasma after intravenous administration of salvianolic acid for injection.

A simple, efficient, and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for simultaneous determination and pharmacokinetic study of salvianolic acid D, rosmarinic acid, lithospermicic acid, salvianolic acid B, and salvianolic acid Y in rat plasma after intravenous administration of salvianolic acid for injection. Three doses of administration, containing 10, 25, and 62.5 mg/kg, were investigated. Plasma samples were pretreated using protein precipitation with pre-cooled acetonitrile. As shown in S1, Chromatographic separation was achieved on a Waters Acquity UPLC® BEH C18 column (1.7 µm, 2.1 × 100 mm) with a mobile phase composed of acetonitrile-methanol-0.5% aqueous formic acid (10:30:60, v/v/v) at a flow rate of 0.3 ml/min. MS was detected by electrospray ion source negative ion mode and multiple reaction monitoring mode. The method was fully validated. The calibration curves for the five phenolic acids were linear in the given concentration ranges. The extraction recoveries, matrix effects, intra-day and inter-day precisions, and accuracies of the five analytes were all within acceptable limits. No significant difference of elimination half-life time (T1/2 ) of five analytes at three doses was observed. Area under the curve and peak concentration (Cmax ) of the five analytes demonstrated a linear increase in the doses with the linear correlation r of each analyte at three doses being greater than 0.915. It indicated that the pharmacokinetic behavior of is positively related to the dose in the range of 10-62.5 mg/kg.

Identification of potential inhibitors of omicron variant of SARS-Cov-2 RBD based virtual screening, MD simulation, and DFT.

Emergence of the SARS-CoV-2 Omicron variant of concern (VOC; B.1.1.529) resulted in a new peak of the COVID-19 pandemic, which called for development of effective therapeutics against the Omicron VOC. The receptor binding domain (RBD) of the spike protein, which is responsible for recognition and binding of the human ACE2 receptor protein, is a potential drug target. Mutations in receptor binding domain of the S-protein have been postulated to enhance the binding strength of the Omicron VOC to host proteins. In this study, bioinformatic analyses were performed to screen for potential therapeutic compounds targeting the omicron VOC. A total of 92,699 compounds were screened from different libraries based on receptor binding domain of the S-protein via docking and binding free energy analysis, yielding the top 5 best hits. Dynamic simulation trajectory analysis and binding free energy decomposition were used to determine the inhibitory mechanism of candidate molecules by focusing on their interactions with recognized residues on receptor binding domain. The ADMET prediction and DFT calculations were conducted to determine the pharmacokinetic parameters and precise chemical properties of the identified molecules. The molecular properties of the identified molecules and their ability to interfere with recognition of the human ACE2 receptors by receptor binding domain suggest that they are potential therapeutic agents for SARS-CoV-2 Omicron VOC.