Synthesis of Poly-γ-Glutamic Acid and Its Application in Biomedical Materials

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TL;DR

This review summarizes the preparation methods of poly-γ-glutamic acid (γ-PGA), including microbial fermentation, chemical synthesis, and enzymatic polymerization, with microbial fermentation being the most scalable and environmentally friendly. γ-PGA hydrogels, crosslinked with materials like chitosan, hyaluronic acid, or gelatin, show promise in drug delivery, tissue engineering, and wound healing due to their biocompatibility, biodegradability, and pH-responsiveness. The clinical significance lies in γ-PGA's potential to improve anti-tumor drug targeting, cartilage repair, and antibacterial wound dressings, though challenges in production stability and clinical validation remain.

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**Background:** Poly-γ-glutamic acid (γ-PGA) is a naturally occurring, biodegradable polymer composed of D- and L-glutamic acid units linked by amide bonds. It was first discovered in the capsule of Bacillus anthracis and later found in Bacillus subtilis, Bacillus natto, and some archaea. γ-PGA exhibits non-cytotoxicity, high water retention, and the ability to bind metal ions via free carboxyl groups. Its molecular weight ranges from 10 to 10,000 kDa, with low-molecular-weight forms used in pharmaceuticals and agriculture, and high-molecular-weight forms enhancing calcium absorption. This review focuses on synthesis methods and medical applications, distinguishing itself from prior reviews that emphasized biosynthesis in microorganisms and agricultural uses.

**Methods:** The review describes four preparation methods for γ-PGA: (1) Chemical synthesis, which involves traditional peptide synthesis or dimer condensation polymerization, but is complex, costly, and polluting, yielding mainly α-PGA. (2) Enzyme conversion using glutamine transpeptidase (GTP) or the RimK enzyme from Escherichia coli, which offers high purity but faces challenges in enzyme isolation and activity. (3) Extraction from natto using organic solvents, which is simple but yields low purity and quantity. (4) Microbial fermentation using strains like Bacillus subtilis and Bacillus licheniformis, which is the predominant industrial method due to its environmental friendliness and scalability. Fermentation can be solid or liquid, with liquid fermentation preferred for better control of pH, temperature, and dissolved oxygen. Strains are categorized as glutamate-dependent (e.g., B. subtilis D7, ZJU-7) or glutamate-independent (e.g., B. subtilis GXG-5). Optimization strategies include metabolic process regulation (e.g., adding Fe²⁺, Na⁺, Ca²⁺, Mn²⁺ ions) and genetic engineering (e.g., overexpressing pgs BCA genes, deleting pgd S and ggt degradation genes). For example, Guo et al. (2023) achieved a γ-PGA yield of 76.848 g/L in a 5 L bioreactor by adding 0.7 g/L FeSO₄·7H₂O and optimizing agitation. Li et al. (2021) increased production by 62% to 12.02 g/L via overexpression of pyruvate dehydrogenase and citrate synthase. Zhu et al. (2022) boosted yield to 26.4 g/L (3.72-fold increase) using crude glycerol and strong promoters.

**Key Results:** γ-PGA is often used as a hydrogel, prepared via physical or chemical crosslinking. Crosslinking with chitosan (CS) yields polyelectrolyte complex (PEC) hydrogels with high tensile strength and pH sensitivity, promoting normal human dermal fibroblast (NHDF) cell proliferation. CS/γ-PGA hydrogels with Dendrobium candidum enzyme (DOE) at 4–6% concentration inhibit Escherichia coli and Staphylococcus aureus. Crosslinking with hyaluronic acid (HA) improves shear resistance; methacrylate-functionalized HA/γ-PGA hydrogels (Ma et al., 2018) showed sustained BSA release over 5 days with >90% cell survival in NIH3T3 fibroblasts. Adaptive HA/γ-PGA hydrogels using Schiff base and photopolymerization (Ma et al., 2020) released 10% of BSA after 60 h and continued for 72 h. Gelatin (GEL)/γ-PGA double-network hydrogels (Dou et al.) maintained >70% light transmittance, crucial for wound observation. In drug delivery, γ-PGA conjugates with paclitaxel (PTX) showed better anti-tumor activity in mouse models (Li et al., 1998). α-PGA-DOX nanoparticles achieved a tumor inhibition rate of 67.4%, 1.5 times higher than DOX injection (Guo et al.). γ-PGA-CA-Pt(IV) prodrugs converted to active platinum in acidic tumor microenvironments. In wound healing, γ-PGA/sericin hydrogels (Shi et al.) and γ-PGA-ε-PL hydrogels (Sun et al.) showed antibacterial effects against Staphylococcus aureus and Escherichia coli. CGLH double-network hydrogel (Hu et al.) promoted infected wound healing within 12 days. In tissue engineering, methacrylate-γ-PGA hydrogels (Yang et al., 2020) aided cartilage reconstruction in rabbit ears. Mussel-inspired bisphosphonated injectable hydrogels (Bo et al.) reduced bone defects in rat skulls.

**Clinical Implications:** γ-PGA-based materials offer significant potential in biomedicine. In oncology, PGA conjugates enhance targeting and reduce side effects of chemotherapeutics like PTX, DOX, and cisplatin. pH-responsive hydrogels enable controlled drug release in tumor microenvironments. In wound healing, γ-PGA dressings provide a moist environment, antibacterial activity, and promote collagen synthesis and cell proliferation, with applications in diabetic wounds and infected wounds. In tissue engineering, γ-PGA hydrogels support cartilage, bone, and neural regeneration due to their high water content, porosity, and biocompatibility. However, challenges include complex production processes, lack of industry standards for medical-grade PGA, and need for extensive clinical validation. Future directions involve optimizing fermentation, genetic engineering for higher yields, and developing composite hydrogels with improved mechanical strength and degradation profiles.