Novel Multitarget ACE Inhibitory Peptides from Bovine Colostrum Immunoglobulin G: Cellular Transport, Efficacy in Regulating Endothelial Dysfunction, and Network Pharmacology Studies.

In this study, we used traditional laboratory methods, bioinformatics, and cellular models to screen novel ACE inhibitory (ACEI) peptides with strong ACEI activity, moderate absorption rates, and multiple targets from bovine colostrum immunoglobulin G (IgG). The purified fraction of the compound proteinase hydrolysate of IgG showed good ACEI activity. After nano-UPLC-MS/MS identification and in silico analysis, eight peptides were synthesized and verified. Among them, SFYPDY, TSFYPDY, FSWF, WYQQVPGSGL, and GVHTFP were identified as ACEI peptides, as they exhibited strong ACEI activity (with IC50 values of 104.7, 80.0, 121.2, 39.8, and 86.3 µM, respectively). They displayed good stability in an in vitro simulated gastrointestinal digestion assay. In a Caco-2 monolayer model, SFYPDY, FSWF, and WYQQVPGSGL exhibited better absorption rates and lower IC50 values than the other peptides and were thereby identified as novel ACEI peptides. Subsequently, in a H2O2-induced endothelial dysfunction (ED) model based on HUVECs, SFYPDY, FSWF, and WYQQVPGSGL regulated ED by reducing apoptosis and ROS accumulation while upregulating NOS3 mRNA expression. Network pharmacology analysis and RT-qPCR confirmed that they regulated multiple targets. Overall, our results suggest that SFYPDY, FSWF, and WYQQVPGSGL can serve as novel multitarget ACEI peptides.

Purification, structural elucidation and biological activities of exopolysaccharide produced by the endophytic Penicillium javanicum from Millettia speciosa Champ.

An acid polysaccharide, named HP, was produced by endophytic Penicillium javanicum MSC-R1 isolated from southern medicine Millettia speciosa Champ. The molecular weight of HP was 37.8 kDa and consisted of Ara f, Galр, Glcр, Manр, and GlcрA with a molar ratio of 1.09: 3.47: 68.48: 16.59: 8.85. The glycosidic linkage of HP was proven to be →3, 4)-α-D-Glcр-(1→6)-α-D-Manр-(1→, →3, 4)-α-D-Glcр-(1→4)-α-D-Glcр-(1→, →3), →6)-α-D-Manр-(1→4)-α-D-Glcр-(1→, →3), ß-D-Galр-(1→3)-α-D-Glcр-(1→, →4), →5)-α-L-Ara f -(1→3)-α-D-Glcр-(1→, →4), →6)-α-D-Manр-(1→4)-α-D-GlcAр-(1→ and →4)-α-D-GlcAр-(1→4)-α-D-Glcр-(1→, →3). Additionally, 250 µg/mL of HP possessed nontoxicity to RAW 264.7 cells and exhibited anti-inflammation activity. HP could significantly restrain the amount of tumor necrosis factor-α, interleukin-6 and NO release in RAW264.7, which property is possibly associated with its abundant glucosidic linkage. These results indicated that HP could be regarded as a ponderable ingredient for the health-beneficial functional foods.

Enhancement of thermostability and catalytic properties of ammonia lyase through disulfide bond construction and backbone cyclization.

Ammonia lyases have great application potential in food and pharmaceuticals owing to their unique ammonia addition reaction and atom economy. A novel methylaspartate ammonia-lyase, EcMAL, from E. coli O157:H7 showed high catalytic activity. To further strengthen its thermostability and activity, disulfide bond and backbone cyclization (cyclase) variants were constructed by rational design, respectively. Among them, variant M3, with a disulfide bond introduced, exhibited a 2.3-fold increase in half-life at 50 °C, while cyclase variant M8 showed better performance, with 25.9-fold increases. The synergistic promotion effect of this combinational strategy on activity and stability was also investigated, and the combined mutant M9 exhibited a 1.1-fold improvement in catalytic efficiency while maintaining good thermostability. Circular dichroism analysis and molecular dynamics simulation confirmed that the main sources of improved thermostability were reduced atomic fluctuation and a more stable secondary structure. To our knowledge, this is the first example of combining the introduction of disulfide bonds with cyclase construction to improve enzyme stability, which was characterized by modification away from the enzyme active center, and provided a new method for adjusting enzyme thermostability.

Structure-guided protein engineering of ammonia lyase for efficient synthesis of sterically bulky unnatural amino acids.

Enzymatic asymmetric amination addition is seen as a promising approach for synthesizing amine derivatives, especially unnatural amino acids, which are valuable precursors to fine chemicals and drugs. Despite the broad substrate spectrum of methylaspartate lyase (MAL), some bulky substrates, such as caffeic acid, cannot be effectively accepted. Herein, we report a group of variants structurally derived from Escherichia coli O157:H7 MAL (EcMAL). A combined mutagenesis strategy was used to simultaneously redesign the key residues of the entrance tunnel and binding pocket to explore the possibility of accepting bulky substrates with potential application to chiral drug synthesis. Libraries of residues capable of lining the active center of EcMAL were then constructed and screened by an effective activity solid-phase color screening method using tyrosinase as a cascade catalyst system. Activity assays and molecular dynamics studies of the resultant variants showed that the substrate specificity of EcMAL was modified by adjusting the polarity of the binding pocket and the degree of flexibility of the entrance tunnel. Compared to M3, the optimal variant M8 was obtained with a 15-fold increase in catalytic activity. This structure-based protein engineering of EcMAL can be used to open new application directions or to develop practical multi-enzymatic processes for the production of various useful compounds.

Immobilization of Cofactor Self-Sufficient Recombinant Escherichia coli for Enantioselective Biosynthesis of (R)-1-Phenyl-1,2-Ethanediol.

(R)-1-phenyl-1,2-ethanediol is an important synthon for the preparation of ß-adrenergic blocking agents. This study identified a (2R,3R)-butanediol dehydrogenase (KgBDH) from Kurthia gibsonii SC0312, which showed high enantioselectivity for production of (R)-1-phenyl-1,2-ethanediol by reduction of 2-hydroxyacetophenone. KgBDH was expressed in a recombinant engineered strain, purified, and characterized. It showed good catalytic activity at pH 6-8 and better stability in alkaline (pH 7.5-8) than an acidic environment (pH 6.0-7.0), providing approximately 73 and 88% of residual activity after 96 h at pH 7.5 and 8.0, respectively. The maximum catalytic activity was obtained at 45°C; nevertheless, poor thermal stability was observed at >30°C. Additionally, the examined metal ions did not activate the catalytic activity of KgBDH. A recombinant Escherichia coli strain coexpressing KgBDH and glucose dehydrogenase (GHD) was constructed and immobilized via entrapment with a mixture of activated carbon and calcium alginate via entrapment. The immobilized cells had 1.8-fold higher catalytic activity than that of cells immobilized by calcium alginate alone. The maximum catalytic activity of the immobilized cells was achieved at pH 7.5, and favorable pH stability was observed at pH 6.0-9.0. Moreover, the immobilized cells showed favorable thermal stability at 25-30°C and better operational stability than free cells, retaining approximately 55% of the initial catalytic activity after four cycles. Finally, 81% yields (195 mM product) and >99% enantiomeric excess (ee) of (R)-1-phenyl-1,2-ethanediol were produced within 12 h through a fed-batch strategy with the immobilized cells (25 mg/ml wet cells) at 35°C and 180 rpm, with a productivity of approximately 54 g/L per day.

Improving the thermostability and activity of Paenibacillus pasadenensis chitinase through semi-rational design.

Chitinase is a promising biocatalyst for chitin biotransformation in the field of recalcitrant biomass degradation. Excellent catalytic performance is conducive to its commercial utilization. In this work, sequence- and structure-based semi-rational design was performed to evolve the thermostability and activity of a previously identified chitinase PpChi1 from Paenibacillus pasadenensis CS0611. After combinational mutagenesis, the mutant S244C-I319C/T259P with disulfide bond introduction and proline substitution exhibited higher specific activity at higher temperature, 26.3-fold in half-life value at 50 °C, and a 7.9 °C rise in half-inactivation temperature T1/215min compared to the wild-type enzyme. The optimal reaction temperature of the mutant was shifted from 45 °C to 52.5 °C. Molecular dynamic simulation and structure analysis confirmed that these improvements of the mutant were attributed to its stabilized folding form, possibly caused by the decreased entropy of unfolding. This work gives an initial insight into the effect of conserved proline residues in thermostable chitinases and proposes a feasible approach for improving chitinase thermostability to facilitate its application in chitin hydrolysis to valuable oligosaccharides.