Whole genome sequencing of the poly-γ-glutamic acid-producing novel Bacillus subtilis Tamang strain, isolated from spontaneously fermented kinema.

Kinema, a traditional fermented soybean food from the Himalayas, is well-liked for its sticky texture and flavourful umami taste. Among 175 bacterial strains from spontaneously fermented kinema samples, Bacillus subtilis Tamang strain stood out for its high stickiness and viscosity. The strain's Poly-γ-glutamic acid (γ-PGA) contains various groups of glutamic acid and has a molecular weight of 660 kDa. It demonstrates the ability to solubilize iron, preserve ferritin in Caco-2 cells, and exhibit antibacterial properties. The genome of B. subtilis Tamang is devoid of plasmid elements but does feature nine insert elements. Noteworthy is the presence of unique secondary metabolites with potential antimicrobial effects, such as amyloliquecidin GF610, bogorol A, and thermoactinoamide A. A total of 132 carbohydrate-active enzymes (CAZy) were identified, hinting at possible prebiotic characteristics. The genome analysis revealed genes responsible for γ-PGA production via the capBCA complex. Furthermore, genes associated with fibrinolytic activity, taste enhancement, biopeptides, immunomodulators, and vitamins like B12 and K2 were found, along with probiotics and various health benefits. The genetic material for L-asparaginase production, known for its anti-cancer properties, was also detected, as well as CRISPR-Cas systems. The absence of virulence factors and antimicrobial resistance genes confirms the safety of consuming B. subtilis Tamang as a food-grade bacterium.

Position Matters: Pyridine Regioisomers Influence Secondary Structure and Micelle Morphology in Polymer-Homopolypeptide Micelles.

Polymer-homopolypeptide block copolymers are a class of bioinspired materials that combine the processability and stability of synthetic polymers with the biocompatibility and unique secondary structures of peptides, such as α-helices and ß-sheets. These properties make them ideal candidates for a wide variety of applications, for example, in the pharmaceutical field, where they are frequently explored as building blocks for polymeric micelle drug delivery systems. While homopolypeptide side chains can be furnished with an array of different moieties to impart the copolymers with desirable properties, such as stimulus responsivity, pyridine derivatives represent an underutilized functional group for this purpose. Additionally, the interplay between polypeptide side chain structure, secondary conformation, and micelle morphology is not yet well understood, particularly in the case of structural regioisomers. Therefore, in this work, a series of polymer-homopolypeptide copolymers were prepared from a poly(ethylene glycol)-b-poly(glutamic acid) (PEG-b-PGA) backbone, where the pendant carboxylic acid groups were covalently conjugated to a series of pyridine regioisomers by carbodiimide coupling. These pyridine regioisomers differed only in the position of the nitrogen heteroatom, ortho, meta or para, relative to the linking group, generating a series of PEG-b-poly(pyridinylmethyl glutamate) (PEG-b-PMG) copolymers. Following self-assembly of the copolymers in aqueous solutions, dynamic light scattering (DLS) revealed differences in micelle hydrodynamic diameter (Dh) (ranging from ∼60 to 120 nm), while transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS) revealed distinctive morphologies ranging from ellipsoidal, to cylindrical, and disc-like, suggesting that subtle changes in positional isomers in the polypeptide block may influence the micelle structure. Analysis of the PEG-b-PMG copolymer micelles by circular dichroism (CD) and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy revealed that differences in the morphology were associated with changes in polypeptide secondary structure, which in turn was influenced by the position of the pyridine heteroatom. Overall, these findings contribute to the broader understanding of the relationship between polypeptide structure and micelle morphology and serve as useful insight for the rational design of polymer-polypeptide nanoparticles.

Heterologous synthesis of poly-γ-glutamic acid enhanced drought resistance in maize (Zea mays L.).

Drought stress is the main factor restricting maize yield. Poly-γ-glutamic acid (γ-PGA), as a water-retaining agent and fertilizer synergist, could significantly improve the drought resistance and yield of many crops. However, its high production costs and unclear long-term impact on soil ecology limit its large-scale application. In this study, an environmentally friendly green material γ-PGA was heterologous synthesized in maize for the first time using the synthetic biology method. The genes (PgsA, PgsB, PgsC) participated in γ-PGA synthesis were cloned from Bacillus licheniformis and transformed into maize to produce γ-PGA for the first time. Under drought stress, transgenic maize significantly increased the ear length, ear weight and grain weight by 50 % compared to the control, whereas the yield characteristic of ear weight, grain number per ear, grain weight per ear and 100-grain weight increased by 1.67 %-2.33 %, 3.78 %-13.06 %, 8.41 %-22.06 %, 6.03 %-19.28 %, and 11.85 %-18.36 %, respectively under normal growth conditions. γ-PGA was mainly expressed in the mesophyll cells of maize leaf rosette structure and improved drought resistance and yield by protecting and increasing the expression of genes for the photosynthetic and carbon fixation. This study is an important exploration for maize drought stress molecular breeding and building resource-saving agriculture.

Cellular pharmacokinetic of methotrexate and its modulation by folylpolyglutamate synthetase and γ-glutamyl hydrolase in tumor cells.

BACKGROUND AND PURPOSE: Clinical studies showed that prolonged infusion of methotrexate (MTX) leads to more severe adverse reactions than short infusion of MTX at the same dose. We hypothesized that it is the saturation of folate polyglutamate synthetase (FPGS) at high MTX concentration that limits the intracellular synthesis rate of methotrexate polyglutamate (MTX-PG). Due to a similar accumulation rate, a longer infusion duration may increase the concentration of MTX-PG and, result in more serious adverse reactions. In this study, we validated this hypothesis. EXPERIMENTAL APPROACH: A549, BEL-7402 and MHCC97H cell lines were treated with MTX at gradient concentrations. Liquid chromatograph-mass spectrometer (UPLC-MS/MS) was used to quantify the intracellular concentration of MTX-PG and the abundance of FPGS and γ-glutamyl hydrolase (GGH). High quality data were used to fit the cell pharmacokinetic model. KEY RESULTS: Both cell growth inhibition rate and intracellular MTX-PG concentration showed a nonlinear relationship with MTX concentration. The parameter Vmax in the model, which represents the synthesis rate of MTX-PG, showed a strong correlation with the abundance of intracellular FPGS. CONCLUSION AND IMPLICATIONS: According to the model fitting results, it was confirmed that the abundance of FPGS is a decisive factor limiting the synthesis rate of MTX-PG. The proposed hypothesis was verified in this study. In addition, based on the intracellular metabolism, a reasonable explanation was provided for the correlation between the severity of adverse reactions of MTX and infusion time. This study provides a new strategy for the individualized treatment and prediction of efficacy/side effects of MTX.

Investigating the role of poly-γ-glutamic acid in Pennisetum giganteum phytoextraction of mercury-contaminated soil.

Farmland mercury (Hg) pollution poses a significant threat to human health, but there is a lack of highly efficient phytoextraction for its remediation at present. This study investigates the impact of poly-γ-glutamic acid (γ-PGA) on the phytoextraction capabilities of Pennisetum giganteum (P. giganteum) in Hg-contaminated soil. Our research indicates that amending γ-PGA to soil markedly enhances the assimilation of soil Hg by P. giganteum and transformation of Hg within itself, with observed increases in Hg concentrations in roots, stems, and leaves by 1.1, 4.3, and 18.9 times, respectively, compared to the control. This enhancement is attributed to that γ-PGA can facilitate the hydrophilic and bioavailable of soil Hg. Besides, γ-PGA can stimulate the abundance of Hg-resistance bacteria Proteobacteria in the rhizosphere of P. giganteum, thus increasing the mobility and uptake of soil Hg by P. giganteum roots. Moreover, the hydrophilic nature of Hg-γ-PGA complexes supports their transport via the apoplastic pathway, across the epidermis, and through the Casparian strip, eventually leading to immobilization in the mesophyll tissues. This study provides novel insights into the mechanisms of Hg phytoextraction, demonstrating that γ-PGA significantly enhances the effectiveness of P. giganteum in Hg uptake and translocation. The findings suggest a promising approach for the remediation of Hg-contaminated soil, offering a sustainable and efficient strategy for environmental management and health risk mitigation.

Construction of Tough Hydrogel Cross-Linked via Ionic Interaction by Protection Effect of Hydrophobic Domains.

Most hydrogels have poor mechanical properties, severely limiting their potential applications, and numerous approaches have been introduced to fabricate more robust and durable examples. However, these systems consist of nonbiodegradable polymers which limit their application in tissue engineering. Herein, we focus on the fabrication and investigate the influence of hydrophobic segments on ionic cross-linking properties for the construction of a tough, biodegradable hydrogel. A biodegradable, poly(γ-glutamic acid) polymer conjugated with a hydrophobic amino acid, l-phenylalanine ethyl ester (Phe), together with an ionic cross-linking group, alendronic acid (Aln) resulting in γ-PGA-Aln-Phe, was initially synthesized. Rheological assessments through time sweep oscillation testing revealed that the presence of hydrophobic domains accelerated gelation. Comparing gels with and without hydrophobic domains, the compressive strength of γ-PGA-Aln-Phe was found to be six times higher and exhibited longer stability properties in ethylenediaminetetraacetic acid solution, lasting for up to a month. Significantly, the contribution of the hydrophobic domains to the mechanical strength and stability of ionic cross-linking properties of the gel was found to be the dominant factor for the fabrication of a tough hydrogel. As a result, this study provides a new strategy for mechanical enhancement and preserves ionic cross-linked sites by the addition of hydrophobic domains. The development of tough, biodegradable hydrogels reported herein will open up new possibilities for applications in the field of biomaterials.

Chitosan nanomedicines-engineered bifidobacteria complexes for effective colorectal tumor-targeted delivery of SN-38.

The clinical application of 7-ethyl hydroxy-camptothecin (SN-38) maintains challenges not only due to its poor solubility and stability but also the lack of effective carriers to actively deliver SN-38 to deep tumor sites. Although SN-38-based nanomedicines could improve the solubility and stability from different aspects, the tumor targeting efficiency remains very low. Leveraging the hypoxic taxis of bifidobacteria bifidum (B. bifi) to the deep tumor area, we report SN-38-based nanomedicines-engineered bifidobacterial complexes for effective tumor-targeted delivery. Firstly, SN-38 was covalently coupled with poly-L-glutamic acid (L-PGA) and obtained soluble polymeric prodrug L-PGA-SN38 to improve its solubility and stability. To prolong the drug release, L-PGA-SN38 was mildly complexed with chitosan to form nanomedicines, and nanomedicines engineered B. bifi were further elaborated via electrostatic interaction of the excess of cationic chitosan shell from nanomedicines and anionic teichoic acid from B. bifi. The engineered B. bifi complexes inherited the bioactivity of native B. bifi and exhibited distinctly enhanced accumulation at the tumor site. More importantly, significantly elevated anti-tumor efficacy was achieved after the treatment of CS-L-PGA-SN38 NPs/B. bifi complexes, with favorable tumor suppression up to 80%. Such a B. bifi-mediated delivery system offers a promising platform for effective drug delivery and enhanced drug accumulation in the hypoxia deep tumor with superior anti-tumor efficacy.

Bionic double-crosslinked hydrogel of poly (γ-glutamic acid)/poly (N-(2-hydroxyethyl) acrylamide) with ultrafast gelling process and ultrahigh burst pressure for emergency rescue.

Injectable adhesive hydrogels combining rapid gelling with robust adhesion to wet tissues are highly required for fast hemostasis in surgical and major trauma scenarios. Inspired by the cross-linking mechanism of mussel adhesion proteins, we developed a bionic double-crosslinked (BDC) hydrogel of poly (γ-glutamic acid) (PGA)/poly (N-(2-hydroxyethyl) acrylamide) (PHEA) fabricated through a combination of photo-initiated radical polymerization and hydrogen bonding cross-linking. The BDC hydrogel exhibited an ultrafast gelling process within 1 s. Its maximum adhesion strength to wet porcine skin reached 254.5 kPa (9 times higher than that of cyanoacrylate (CA) glue) and could withstand an ultrahigh burst pressure of 626.4 mmHg (24 times higher than that of CA glue). Notably, the BDC hydrogel could stop bleeding within 10 s from a rat liver incision 10 mm long and 5 mm deep. The wound treated with the BDC hydrogel healed faster than the control groups, underlining the potential for emergency rescue and wound care scenarios.

The biological response of pH-switch-based gold nanoparticle-composite polyamino acid embolic material.

With continuous advances in medical technology, non-invasive embolization has emerged as a minimally invasive treatment, offering new possibilities in cancer therapy. Fluorescent labeling can achieve visualization of therapeutic agents in vivo, providing technical support for precise treatment. This paper introduces a novel in situ non-invasive embolization composite material, Au NPs@(mPEG-PLGTs), created through the electrostatic combination of L-cysteine-modified gold nanoparticles (Au NPs) and methoxy polyethylene glycol amine-poly[(L-glutamic acid)-(L-tyrosine)] (mPEG-PLGTs). Experiments were undertaken to confirm the biocompatibility, degradability, stability and performance of this tumor therapy. The research results demonstrated a reduction in tumor size as early as the fifth day after the initial injection, with a significant 90% shrinkage in tumor volume observed after a 20-day treatment cycle, successfully inhibiting tumor growth and exhibiting excellent anti-tumor effects. Utilizing near-infrared in vivo imaging, Au NPs@(mPEG-PLGTs) displayed effective fluorescence tracking within the bodies of nude BALB-c mice. This study provides a novel direction for the further development and innovation of in situ non-invasive embolization in the field, highlighting its potential for rapid, significant therapeutic effects with minimal invasiveness and enhanced safety.

Injectable polyamide-amine dendrimer-crosslinked meloxicam-containing poly-γ-glutamic acid hydrogel for prevention of postoperative tissue adhesion through inhibiting inflammatory responses and balancing the fibrinolytic system.

Establishing a physical barrier between the peritoneum and the cecum is an effective method to reduce the risk of postoperative abdominal adhesions. Meloxicam (MX), a nonsteroidal anti-inflammatory drug has also been applied to prevent postoperative adhesions. However, its poor water solubility has led to low bioavailability. Herein, we developed an injectable hydrogel as a barrier and drug carrier for simultaneous postoperative adhesion prevention and treatment. A third-generation polyamide-amine dendrimer (G3) was exploited to dynamically combine with MX to increase the solubility and the bioavailability. The formed G3@MX was further used to crosslink with poly-γ-glutamic acid (γ-PGA) to prepare a hydrogel (GP@MX hydrogel) through the amide bonding. In vitro and in vivo experiments evidenced that the hydrogel had good biosafety and biodegradability. More importantly, the prepared hydrogel could control the release of MX, and the released MX is able to inhibit inflammatory responses and balance the fibrinolytic system in the injury tissues in vivo. The tunable rheological and mechanical properties (compressive moduli: from âˆ¼ 57.31 kPa to âˆ¼ 98.68 kPa;) and high anti-oxidant capacity (total free radical scavenging rate of âˆ¼ 94.56 %), in conjunction with their syringeability and biocompatibility, indicate possible opportunities for the development of advanced hydrogels for postoperative tissue adhesions management.

Enhancing Poly-γ-glutamic Acid Production in Bacillus tequilensis BL01 through a Multienzyme Assembly Strategy and Expression Features of Glutamate Synthesis from Corynebacterium glutamicum.

The enhancement of intracellular glutamate synthesis in glutamate-independent poly-γ-glutamic acid (γ-PGA)-producing strains is an essential strategy for improving γ-PGA production. Bacillus tequilensis BL01ΔpgdSΔggtΔsucAΔgudB:P43-ppc-pyk-gdhA for the efficient synthesis of γ-PGA was constructed through expression of glutamate synthesis features of Corynebacterium glutamicum, which increased the titer of γ-PGA by 2.18-fold (3.24 ± 0.22 g/L) compared to that of B. tequilensis BL01ΔpgdSΔggtΔsucAΔgudB (1.02 ± 0.11 g/L). To further improve the titer of γ-PGA and decrease the production of byproducts, three enzymes (Ppc, Pyk, and AceE) were assembled to a complex using SpyTag/Catcher pairs. The results showed that the γ-PGA titer of the assembled strain was 31.31% higher than that of the unassembled strain. To further reduce the production cost, 25.73 ± 0.69 g/L γ-PGA with a productivity of 0.48 g/L/h was obtained from cheap molasses. This work provides new metabolic engineering strategies to improve the production of γ-PGA in B. tequilensis BL01. Furthermore, the engineered strain has great potential for the industrial production of γ-PGA from molasses.

Production of ultra-high-molecular-weight poly-γ-glutamic acid by a newly isolated Bacillus subtilis strain and genomic and transcriptomic analyses.

Poly-γ-glutamic acid (γ-PGA) is a microbial-derived polymer with molecular weight (Mw) from 104 to 107 Da, and the high-Mw (> 7.0 × 105 Da) or ultra-high-Mw (> 5.0 × 106 Da) γ-PGA has important application value as a tissue engineering material, as a flocculant, and as a heavy metal remover. Therefore, how to produce these high-Mw γ-PGAs with low cost and high efficiency has attracted wide attention. In this study, a γ-PGA producer was isolated from the natural environment, and identified and named Bacillus subtilis GXD-20. Then, the ultra-high-Mw (> 6.0 × 106 Da) γ-PGA produced by GXD-20 was characterized. Interestingly, GXD-20 could produce γ-PGA at 42°C, and exhibited a γ-PGA titer of up to 22.29 ± 0.59 g L-1 in a 5-L fermenter after optimization of the fermentation process. Comparative genomic analysis indicated that the specific protein sequence and subcellular localization of PgdS (a γ-PGA-degrading enzyme) were closely related to the ultra-high-Mw of γ-PGA. Transcriptomic analysis revealed that the high γ-PGA titer at 42°C was mainly related to the high expression of genes encoding enzymes for sucrose transportation and utilization, nitrogen transportation, endogenous glutamate synthesis, and γ-PGA synthesis. These results provide new insights into the production of ultra-high-Mw γ-PGA by Bacillus at high temperatures.

Layer-by-layer assembly of quercetin-loaded zein/γPGA/low-molecular-weight chitosan/fucoidan nanosystem for targeting inflamed blood vessels.

Chitosan acts as a versatile carrier in polymeric nanoparticle (NP) for diverse drug administration routes. Delivery of antioxidants, such as quercetin (Qu) showcases potent antioxidant and anti-inflammatory properties for reduction of various cardiovascular diseases, but low water solubility limits uptake. To address this, we developed a novel layer-by-layer zein/gamma-polyglutamic acid (γPGA)/low-molecular-weight chitosan (LC)/fucoidan NP for encapsulating Qu and targeting inflamed vessel endothelial cells. We used zein (Z) and γPGA (r) to encapsulate Qu (Qu-Zr NP) exhibited notably higher encapsulation efficiency compared to zein alone. Qu-Zr NP coated with LC (Qu-ZrLC2 NP) shows a lower particle size (193.2 ± 2.9 nm), and a higher zeta potential value (35.2 ± 0.4 mV) by zeta potential and transmission electron microscopy analysis. After coating Qu-ZrLC2 NP with fucoidan, Qu-ZrLC2Fa NP presented particle size (225.16 ± 0.92 nm), zeta potential (-25.66 ± 0.51 mV) and maintained antioxidant activity. Further analysis revealed that Qu-ZrLC2Fa NP were targeted and taken up by HUVEC cells and EA.hy926 endothelial cells. Notably, we observed Qu-ZrLC2Fa NP targeting zebrafish vessels and isoproterenol-induced inflamed vessels of rat. Our layer-by-layer formulated zein/γPGA/LC/fucoidan NP show promise as a targeted delivery system for water-insoluble drugs. Qu-ZrLC2Fa NP exhibit potential as an anti-inflammatory therapeutic for blood vessels.

Balancing mechanical property and swelling behavior of bacterial cellulose film by in-situ adding chitosan oligosaccharide and covalent crosslinking with γ-PGA.

Bacterial cellulose (BC) is an ideal candidate material for drug delivery, but the disbalance between the swelling behavior and mechanical properties limits its application. In this work, covalent crosslinking of γ-polyglutamic acid (γ-PGA) with the chitosan oligosaccharide (COS) embedded in BC was designed to remove the limitation. As a result, the dosage, time, and batch of COS addition significantly affected the mechanical properties and the yield of bacterial cellulose complex film (BCCF). The addition of 2.25 % COS at the incubation time of 0.5, 1.5, and 2 d increased the Young's modulus and the yield by 5.65 and 1.42 times, respectively, but decreased the swelling behavior to 1774 %, 46 % of that of native BC. Covalent γ-PGA transformed the dendritic structure of BCCF into a spider network, decreasing the porosity and increasing the swelling behavior by 3.46 times. The strategy balanced the swelling behavior and mechanical properties through tunning hydrogen bond, electrostatic interaction, and amido bond. The modified BCCF exhibited a desired behavior of benzalkonium chlorides transport, competent for drug delivery. Thereby, the strategy will be a competent candidate to modify BC for such potential applications as wound dressing, artificial skin, scar-inhibiting patch, and so on.

Enhanced environmental stress resistance and functional properties of the curcumin-shellac nano-delivery system: Anti-flocculation of poly-γ-glutamic acid.

Curcumin was widely designed as nanoparticles to remove application restrictions. The occurrence of flocculation is a primary factor limiting the application of the curcumin nano-delivery system. To enhance the environmental stress resistance and functional properties of shellac-curcumin nanoparticles (S-Cur-NPs), γ-polyglutamic acid (γ-PGA) was utilized as an anti-flocculant. The encapsulation efficiency and loading capacity of S-Cur-NPs were also improved with γ-PGA incorporation. FTIR and XRD analysis confirmed the presence of amorphous characteristics in S-Cur-NPs and the combination of γ-PGA and shellac was driven by hydrogen bonding. The hydrophilic, thermodynamic, and surface potential of S-Cur-NPs was improved by the incorporation of γ-PGA. This contribution of γ-PGA on S-Cur-NPs effectively mitigated the flocculation occurrence during heating, storage, and in-vitro digestive treatment. Furthermore, it was revealed that γ-PGA enhanced the antibacterial and antioxidant properties of S-Cur-NPs and effectively protected the functional activity against heating, storage, and in-vitro digestion. Release studies conducted in simulated gastrointestinal fluids revealed that S-Cur-NPs have targeted intestinal release properties. Overall, the design of shellac with γ-PGA was a promising strategy to relieve the application stress of shellac and curcumin in the food industry.

Chitosan oligosaccharide-functionalized nano-prodrug for cascade chemotherapy through oxidative stress amplification.

Redox nanoparticles have been extensively developed for chemotherapy. However, the intracellular oxidative stress induced by constant aberrant glutathione (GSH), reactive oxygen species (ROS) and gamma-glutamyl transpeptidase (GGT) homeostasis remains the primary cause of evading tumor apoptosis. Herein, an oxidative stress-amplification strategy was designed using a pH-GSH-H2O2-GGT sensitive nano-prodrug for precise synergistic chemotherapy. The disulfide bond- conjugated doxorubicin prodrug (DOX-ss) was constructed as a GSH-scavenger. Then, phenylboronic acid (PBA), DOX-ss and poly (γ-glutamic acid) (γ-PGA) were successively conjugated using chitosan oligosaccharide (COS) to obtain the nano-prodrug PBA-COS-ss-DOX/γ-PGA. The PBA-COS-ss-DOX/γ-PGA prodrug could tightly attach to the polymer chain segment by atom transfer radical polymerization. Simultaneously, the drug interacted relatively weakly with the polymer by encapsulating ionic crosslinkers in DOX@PBA-COS/γ-PGA. The disulfide bond of the DOX-ss prodrug as a GSH-scavenger could be activated using overexpressed GSH to release DOX. Particularly, PBA-COS-ss-DOX/γ-PGA could prevent premature drug leakage and facilitate DOX delivery by GGT-targeting and intracellular H2O2-cleavable linker in human hepatocellular carcinoma (HepG2) cells. Concurrently, the nano-prodrug induced strong oxidative stress and tumor cell apoptosis. Collectively, the pH-GSH-H2O2-GGT responsive nano-prodrug shows potential for synergistic tumor therapy.

Genomic characterization and related functional genes of γ- poly glutamic acid producing Bacillus subtilis.

γ- poly glutamic acid (γ-PGA), a high molecular weight polymer, is synthesized by microorganisms and secreted into the extracellular space. Due to its excellent performance, γ-PGA has been widely used in various fields, including food, biomedical and environmental fields. In this study, we screened natto samples for two strains of Bacillus subtilis N3378-2at and N3378-3At that produce γ-PGA. We then identified the γ-PGA synthetase gene cluster (PgsB, PgsC, PgsA, YwtC and PgdS), glutamate racemase RacE, phage-derived γ-PGA hydrolase (PghB and PghC) and exo-γ-glutamyl peptidase (GGT) from the genome of these strains. Based on these γ-PGA-related protein sequences from isolated Bacillus subtilis and 181 B. subtilis obtained from GenBank, we carried out genotyping analysis and classified them into types 1-5. Since we found B. amyloliquefaciens LL3 can produce γ-PGA, we obtained the B. velezensis and B. amyloliquefaciens strains from GenBank and classified them into types 6 and 7 based on LL3. Finally, we constructed evolutionary trees for these protein sequences. This study analyzed the distribution of γ-PGA-related protein sequences in the genomes of B. subtilis, B. velezensis and B. amyloliquefaciens strains, then the evolutionary diversity of these protein sequences was analyzed, which provided novel information for the development and utilization of γ-PGA-producing strains.

Enzyme-Triggered Polyelectrolyte Complex for Responsive Delivery of α-Helical Polypeptides to Optimize Antibacterial Therapy.

Responsive nanomaterials hold significant promise in the treatment of bacterial infections by recognizing internal or external stimuli to achieve stimuli-responsive behavior. In this study, we present an enzyme-responsive polyelectrolyte complex micelles (PTPMN) with α-helical cationic polypeptide as a coacervate-core for the treatment of Escherichia coli (E. coli) infection. The complex was constructed through electrostatic interaction between cationic poly(glutamic acid) derivatives and phosphorylation-modified poly(ethylene glycol)-b-poly(tyrosine) (PEG-b-PPTyr) by directly dissolving them in aqueous solution. The cationic polypeptide adopted α-helical structure and demonstrated excellent broad-spectrum antibacterial activity against both Gram-negative and Gram-positive bacteria, with a minimum inhibitory concentration (MIC) as low as 12.5 µg mL-1 against E. coli. By complexing with anionic PEG-b-PPTyr, the obtained complex formed ß-sheet structures and exhibited good biocompatibility and low hemolysis. When incubated in a bacterial environment, the complex cleaved its phosphate groups triggered by phosphatases secreted by bacteria, exposing the highly α-helical conformation and restoring its effective bactericidal ability. In vivo experiments confirmed accelerated healing in E. coli-infected wounds.

Toward Synthetic Intrinsically Disordered Polypeptides (IDPs): Controlled Incorporation of Glycine in the Ring-Opening Polymerization of N-Carboxyanhydrides.

Intrinsically disordered proteins (IDPs) do not have a well-defined folded structure but instead behave as extended polymer chains in solution. Many IDPs are rich in glycine residues, which create steric barriers to secondary structuring and protein folding. Inspired by this feature, we have studied how the introduction of glycine residues influences the secondary structure of a model polypeptide, poly(l-glutamic acid), a helical polymer. For this purpose, we carried out ring-opening copolymerization with γ-benzyl-l-glutamate and glycine N-carboxyanhydride (NCA) monomers. We aimed to control the glycine distribution within PBLG by adjusting the reactivity ratios of the two NCAs using different reaction conditions (temperature, solvent). The relationship between those conditions, the monomer distributions, and the secondary structure enabled the design of intrinsically disordered polypeptides when a highly gradient microstructure was achieved in DMSO.

Layer-by-layer self-assembly embedding of nattokinase in chitosan/γ-polyglutamic acid: Preparation, fibrinolytic activity, stability, and in vitro digestion study.

Nattokinase (NK) is a thrombolytic enzyme extracted from natto, which can be used to prevent and treat blood clots. However, it is sensitive to the environment, especially the acidic environment of human stomach acid, and its effect of oral ingestion is minimal. This study aims to increase NK's oral and storage stability by embedding NK in microcapsules prepared with chitosan (CS) and γ-polyglutamic acid (γ-PGA). The paper prepared a double-layer NK oral delivery system by layer self-assembly and characterized its stability and in vitro simulated digestion. According to the research results, the bilayer putamen structure has a protective effect on NK, which not only maintains high activity in various environments (such as acid-base, high temperature) and long-term storage (60 days), but also effectively protects the loaded NK from being destroyed in gastric fluid and achieves its slow release. This work has proved the feasibility of the design of bilayer putamen structure in oral administration and has good fibrolytic activity. Therefore, the novel CS/γ-PGA microcapsules are expected to be used in nutraceutical delivery systems.