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Poly-γ-glutamic acid (γ-PGA) is a novel biodegradable polyamide material. Microbial fermentation is the only way to produce γ-PGA, but the molecular weight of γ-PGA varied depending on different strains and culture conditions used. The molecular weight of γ-PGA is a main factor affecting the utilization of γ-PGA. It is urgent to find an efficient way to prepare γ-PGA with specific molecular weight, especially low molecular weight. Bacillus subtilis ECUST is a glutamate-dependent strain that produces γ-PGA. In this study, a recombinant B. subtilis harboring the γ-PGA synthase gene cluster pgs BCAE of our preciously identified γ-PGA–producing B. subtilis ECUST was constructed. Assay of γ-PGA contents and properties showed that recombinant B. subtilis 1A751-pBNS2- pgs BCAE obtained the ability to synthesize γ-PGA with low molecular weight (about 10 kDa). The excessive addition of glutamate inhibited the γ-PGA synthesis, while the addition of Zn 2+ could promote the synthesis of γ-PGA by increasing the transcription of pgs B but had no effect on the molecular weight of synthesized γ-PGA. Under optimized conditions, γ-PGA produced by recombinant B. subtilis 1A751-pBNS2- pgs BCAE increased from initial 0.54 g/L to 3.9 g/L, and the glutamate conversion rate reached 78%. Recombinant B. subtilis 1A751-pBNS2- pgs BCAE has the potential for efficient preparation of low molecular weight γ-PGA.

Chronic trauma repair is an important issue affecting people's healthy lives. Thermo-sensitive hydrogel is injectable in situ and can be used to treat large-area wounds. In addition, antioxidants play important roles in promoting wound repair. The purpose of this research was to prepare a novel thermo-sensitive hydrogel-poly(N-isopropyl-acrylamide)/poly(γ-glutamic acid) (PP) loaded with superoxide dismutase (SOD) to improve the effect for trauma treatment. The micromorphology of the hydrogel was observed by scanning electron microscope and the physical properties were measured. The biocompatibility of hydrogel was evaluated by MTT experiment, and the effect of hydrogel on skin wound healing was evaluated by in vivo histological staining. Gelling behavior and differential scanning calorimeter outcomes showed that the PP hydrogels possessed thermo-sensitivity at physiological temperature and the phase transformation temperature was 28.2°C. The high swelling rate and good water retention were conducive to wound healing. The activity of SOD in vitro was up to 85% at 10 h, which was advantageous to eliminate the superoxide anion. MTT assay revealed that this hydrogel possessed good biocompatibility. Dressings of PP loaded with SOD (SOD-PP) had a higher wound closure rate than other treatments in vivo in diabetic rat model. The SOD-PP thermo-sensitive hydrogels can effectively promote wound healing and have good application prospects for wound repair.

Triptolide (TP) displays a strong immunosuppression function in immune-mediated diseases, especially in the treatment of rheumatoid arthritis. However, in addition to its medical and health-related functions, TP also exhibits diverse pharmacological side effects, for instance, liver and kidney toxicity and myelosuppression. In order to reduce the side effects, a nano drug carrier system (γ-PGA-l-PAE-TP [PPT]), in which TP was loaded by a poly-γ-glutamic acid-grafted l-phenylalanine ethylester copolymer, was developed. PPT was characterized by photon scattering correlation spectroscopy and transmission electron microscopy, which demonstrated that the average diameter of the drug carrier system is 98±15 nm, the polydispersity index is 0.18, the zeta potential is -35 mV, and the TP encapsulation efficiency is 48.6% with a controlled release manner. The methylthiazolyldiphenyl-tetrazolium bromide assay and flow cytometry revealed that PPT could decrease toxicity and apoptosis induced by free TP on RAW264.7 cells, respectively. The detection of reactive oxygen species showed that PPT could decrease the cellular reactive oxygen species induced by TP. Compared with the free TP-treated group, PPT improved the survival rate of the mice (P<0.01) and had no side effects or toxic effects on the thymus index (P>0.05) and spleen index (P>0.05). The blood biochemical indexes revealed that PPT did not cause much damage to the kidney (blood urea nitrogen and creatinine), liver (serum alanine aminotransferase and aspartate aminotransferase), or blood cells (P>0.05). Meanwhile, hematoxylin and eosin staining and terminal-deoxynucleotidyl transferase dUTP nick-end labeling staining indicated that PPT reduced the damage of free TP on the liver, kidney, and spleen. Our results demonstrated that PPT reduced free TP toxicity in vitro and in vivo and that it is a promising fundamental drug delivery system for rheumatoid arthritis treatment.

Background Poly-γ-glutamic acid (γ-PGA) is biodegradable, water-soluble, environment-friendly, and edible. Consequently, it has a variety of industrial applications. It is crucial to control production cost and increase output for industrial production γ-PGA. Results Here γ-PGA production from sugarcane molasses by Bacillus licheniformis CGMCC NO. 23967 was studied in shake-flasks and bioreactors, the results indicate that the yield of γ-PGA could reach 40.668 g/L in a 5L stirred tank fermenter. Further study found that γ-PGA production reached 70.436 g/L, γ-PGA production and cell growth increased by 73.20% and 55.44%, respectively, after FeSO 4 ·7H 2 O was added. Therefore, we investigated the metabolomic and transcriptomic changes following FeSO 4 ·7H 2 O addition. This addition resulted in increased abundance of intracellular metabolites, including amino acids, organic acids, and key TCA cycle intermediates, as well as upregulation of the glycolysis pathway and TCA cycle. Conclusions These results compare favorably with those obtained from glucose and other forms of biomass feedstock, confirming that sugarcane molasses can be used as an economical substrate without any pretreatment. The addition of FeSO 4 ·7H 2 O to sugarcane molasses may increase the efficiency of γ-PGA production in intracellular.

Poly-γ‑glutamic acid (γ‑PGA) produced by Bacillus species is a natural biopolymer, which is widely used in various fields including food, pharmaceuticals, and cosmetics. In this study, the screening of 19 Bacillus isolates derived from traditionally fermented foods revealed that Bacillus velezensis VCN56 was the most potent γ‑PGA producer. The maximum concentration of crude γ‑PGA was 32.9 ± 1.5 g/L in the PGA-3 medium containing glycerol, citric acid, sodium glutamate, NH4Cl, and starch. The resulting γ-PGA was purified and then characterized by HPLC, FTIR, and 1H-NMR analyses. Molecular weight of purified γ‑PGA was estimated to be 98 kDa with a polydisperse index of 2.04. Notably, the pure γ‑PGA showed significant in vitro antioxidant scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (72.0 ± 1.5%), hydroxyl (81.0 ± 0.6%), and superoxide (43.9 ± 0.8%) radicals at the concentration of 4 mg/mL. Using whole-genome sequencing, the genetic organization of pgs operon responsible for γ‑PGA biosynthesis in B. velezensis VCN56 differs from those in other Bacillus genomes. Further genome analysis revealed metabolic pathways for γ-PGA production and degradation. For the first time, the present study provides a better understanding of γ-PGA with a promising antioxidant activity produced by B. velezensis at the phenotypic, biochemical, and genomic levels, which hold potential applications in the foods, cosmetics, and pharmaceutical industries.

In this study, a farmland contaminated by heavy metals (Cu, Zn, Ni, and Cr) was selected to evaluate the performance of poly-γ-glutamic acid (γ-PGA) on the removal of heavy metals in soil washing. The highest heavy metal concentrations at the contaminated site were Cu: 1180 mg/kg, Zn: 1450 mg/kg, Ni: 287 mg/kg, and Cr: 316 mg/kg. Batch experiments designed by Taguchi Method were conducted first to assess the effect of different washing conditions on the removal of heavy metals in laboratory. The results of batch experiments show that factors that affected the removal efficiency of heavy metals was of the order γ-PGA concentration > washing time > liquid/soil ratio > rotational speed. The optimal operating parameters for heavy metal removal were γ-PGA 3.5%, liquid/soil ratio 15/1, washing time 60 min, and rotational speed 100 rpm. Under the optimal conditions, up to 50.7% of the major target metal, Cu, was removed. Heavy metals in the soil were mainly Fe-Mn oxide bound and organically bound. On-site treatment using the optimal operating parameters caused 54.3% of Cu removal. When the soil was washed 3 times by γ-PGA, the removal efficiency of Cu was improved to 74.3%. After the treatment, the change in soil bacterial number was insignificant, indicating that γ-PGA is an environmentally friendly washing reagent.

The presented work discusses the highly efficient esterification of poly (γ-glutamic acid) (γ-PGA) with alkyl halides at room temperature. The esterification reaction was completed within 3 h, and the prepared γ-PGA esters were obtained with excellent yields (98.6%) when 1,1,3,3-tetramethylguanidine (TMG) was used as a promoter. The influence of the amount of TMG, solvent, reaction conditions, and alkyl halides on the esterification reaction was examined. It was found that polar aprotic solvents, such as N-Methylpyrrolidone (NMP) and 1,3-Dimethyl-2-imidazolidinone (DMI), were favorable for the esterification. Non-polar or weakly polar solvents (i.e., dichloroethane, acetonitrile) were not favorable for the esterification. Water as a solvent had a negative effect on esterification. The reactivity of bromine halogenated compounds was higher than that of chlorine halogenated compounds but lower than that of iodine halogenated compounds. The structures of the prepared γ-PGA ester were confirmed by 1H NMR and FT-IR spectroscopy. Thermal stability and hydrophobic properties of the resulting product were tested. The results showed that the prepared γ-PGA propyl ester had high thermal stability (up to 267 °C) and showed good hydrophobicity (contact angle 118.7°).

The aim of this study is to enhance the stability of zein nanoparticles by using poly(γ‐glutamic acid) (γ‐PGA) as a stabilizer. Zein/γ‐PGA nanocomposite particles are produced through a straightforward anti‐solvent precipitation method. The incorporation of γ‐PGA influenced the average particle size, zeta potential, and overall stability of the resulting zein/γ‐PGA nanocomposite particles. These particles exhibit greater resistance to aggregation and sedimentation compared to zein nanoparticles across various environmental conditions, including a wide pH range (3.0–9.0), elevated temperatures (80 °C for 120 min), high ionic strength (1000 mm), and prolonged storage at 4 °C (up to 3 months). Fluorescence spectroscopy reveals significant interactions between zein and γ‐PGA. Fourier transform infrared spectroscopy and zeta potential measurements indicate that hydrogen bonding, hydrophobic interactions, and electrostatic attraction are the primary mechanisms driving these interactions. Importantly, the conditions for forming zein/γ‐PGA nanocomposite particles are effectively utilized to encapsulate a hydrophobic bioactive model (curcumin) with high encapsulation efficiency. The encapsulated curcumin demonstrates improved stability and an amorphous structure compared to free curcumin. Based on these results, zein/γ‐PGA nanocomposite particles can serve as a promising vehicle for hydrophobic active ingredients in food, pharmaceuticals, and cosmetics. Biocompatible zein/poly(γ‐glutamic acid) nanocomposite particles are developed to enhance the stability of zein nanoparticles, which demonstrate excellent stability under a variety of environmental conditions and effective thermal protection for the encapsulated actives.

Agricultural wastes rich in lignocellulosic biomass have been used in the production of poly-γ-glutamic acid (γ-PGA) through separate hydrolysis and fermentation (SHF), but this process is complicated and generates a lot of wastes. In order to find a simpler and greener way to produce γ-PGA using agricultural wastes, this study attempted to establish simultaneous saccharification and fermentation (SSF) with citric acid-pretreated corn straw. The possibility of Bacillus amyloliquefaciens JX-6 using corn straw as substrate to synthesize γ-PGA was validated, and the results showed that increasing the proportion of glucose in the substrate could improve the γ-PGA yield. Based on these preliminary results, the corn straw was pretreated using citric acid. Then, the liquid fraction (xylan-rich) was used for cultivation of seed culture, and the solid fraction (glucan-rich) was used as the substrate for SSF. In a 10-L fermenter, the maximum cumulative γ-PGA concentration in batch and fed-batch SSF were 5.08 ± 0.78 g/L and 10.78 ± 0.32 g/L, respectively. Moreover, the product from SSF without γ-PGA extraction was used as a fertilizer synergist, increasing the yield of pepper by 13.46% (P < 0.05). Our study greatly simplified the production steps of γ-PGA, and each step achieved zero emission as far as possible. The SSF process for γ-PGA production provided a simple and green way for lignocellulose biorefinery and sustainable cultivation in agricultureGraphic abstract

Soil acidification is a worldwide eco-environmental problem detrimental to plant growth and threatening food security. In this study, calcium poly(aspartic acid) (PASP-Ca) and calcium poly-γ-glutamic acid (γ-PGA-Ca) were obtained through cation exchange and used to mitigate soil acidity owing to high solubility and complexing capability. Three rates at 6.7, 13.4, and 20.1 g kg −1 , denoted as PASP-Ca1, PASP-Ca2, and PASP-Ca3, and γ-PGA-Ca (7.4 g kg −1 ) were surface-applied and compared with conventional lime (CaCO 3 , 2.5 g kg −1 ) along with control in two soil layers (top soil 0–10 cm, subsoil 10–20 cm). After leaching, various soil properties and aluminum fractions were measured to assess their ameliorative performance and mechanisms. Although lime achieved the highest soil pH (6.91) in the topsoil followed by PASP-Ca and γ-PGA-Ca (pH: 5.57–6.33), it had less effect on subsoil increase (5.3) vs. PASP-Ca and γ-PGA-Ca (pH: 5.44–5.74). Surface-applied PASP-Ca demonstrated efficiency in elevating soil pH and reducing exchangeable acidity, mainly as exchangeable Al 3+ , whereas γ-PGA-Ca addition superiorly improved soil pH buffering capacity (pHBC). Moreover, PASP-Ca and γ-PGA-Ca addition improved organic carbon by 34.4–44.9%, available P by 4.80–20.71%, and cation exchange capacity (CEC) by 6.19–29.2%, thus greatly enhanced soil fertility. Ca 2+ from polyAA-Ca promoted the displacement of exchangeable Al 3+ or H + from soil colloid, which were subsequently complexed or protonated and facilitated leaching. Additionally, the transformation into stable organo-aluminum fractions via complexation inhibited further hydrolysis. Under PASP-Ca or γ-PGA-Ca addition, the saturation of aluminum in cation exchange complex was reduced 2.91–7.81% compared to the control without addition amendments. Thus, PASP-Ca and γ-PGA-Ca can serve as potent ameliorants to alleviate soil acidity and aluminum toxicity for sustainable agricultural development.