Microbial Poly-(glutamic acid): Production, Biosynthesis, Properties, and Their Applications in the Food and Biomedicals

1. Introduction

Biopolymers, defined as naturally occurring substances or polymeric biomolecules synthesized by living organisms during their entire life cycle, have emerged as a promising alternative to their synthetic counterparts. These biopolymers, categorized into three distinct classes i.e., polypeptide, polysaccharide, and polynucleotide based on their monomeric units, possess remarkable versatility and can be easily manipulated and modified to suit a wide range of applications [1].

The natural process of food and beverage fermentation involves the presence of both beneficial and non-beneficial microorganisms. This results in the product changing its composition, which can lead to improved access to nutrients, the breakdown of toxins, and the reduction of anti-nutritional components. The fermentation of whole cereal products has been linked to various functional benefits, including the reduction of cholesterol levels, enhancement of antioxidant and anti-inflammatory properties, and improvement in vascular function [2]. Depending on the fermentation condition and food substrate, various bioactive metabolites including biopolymers such as polyhydroxyalkanoates, polylactic acid, and polyglutamic acid produced by non-infectious Bacillus and Lactobacillus spp. strains [3]. Polypeptides (poly amino acids) that are produced naturally are of various kinds such as poly-γ-glutamic acid (γ-PGA), poly-ε-lysine (ε-PL), and cyanophycin [4]. γ-PGA an anionic, water-soluble, biodegradable, non-toxic extra-cellular viscous material is produced predominantly by Bacillus strains.

γ-PGA was discovered by Ivanovics as a Bacillus anthracis capsule, while autoclaving the aged and autolyzed cells, γ-PGA was released into the medium [5]. This edible biopolymer consists of D- and L-glutamic acid residues and has diverse applications as humectants, thickeners, cryoprotectants, edible films, heavy metal absorbents, drug carriers, and biological adhesives. Owing to its outstanding water solubility, biodegradability, edibility, and non-toxic nature, γ-PGA and its derivatives have found extensive use in various industrial sectors, including food and pharmaceutical industries [6,7].

Unlike other proteins and peptides that are normally composed of α-amino and α-carboxylic acid units which are prone to protease digestion, γ-PGA differs from them by possessing γ-amide linkages (α-amino and γ-carboxylic units) which prevents its degradation by the action of proteases [8]. Cost-effective substrates and efficient strains are required for the production of the biopolymers commercially through microbial fermentation [9]. Although α-PGA can also be synthesized chemically and through biotransformation these ways are neither economic nor ecological [10].

The preference for L-amino acids in cellular protein synthesis results in the production of proteins that lack D-amino acids. This homochirality is also reflected in the stereochemistry of γ-PGA, where the arrangement of D- and L- forms of glutamic acid is crucial [11]. The molecular weight of γ-PGA, which can range from 100 to 2500 kDa, significantly affects its chain length; as the molecular weight increases, so does the viscosity of the biopolymer, indicating a direct correlation between these two properties [12]. Furthermore, the stability of the α-helical conformation of γ-PGA is maintained within a pH range of 2.5 to 5.5, facilitating the formation of additional COO^- groups [13] in its side chains. An increase in pH beyond 5.5 results in the formation of aggregates, which can lead to arrangements resembling amyloid fibrils [14,15].

Biopolymers have been greatly gaining interest in polymer industries due to their non-chemical production. It is a direct yes to the industries looking for eco-friendly and green synthesis alternatives. Polymers produced biologically are not only environment-friendly ways over chemical-based polymers but are also easy to maintain and produce, and above all they are cost-effective. The gamma-polyglutamic acid and its derivatives have become eminent biopolymers due to their widespread applications in the fields of food to drug discovery [12]. The good physico-chemical properties like water solubility, water-holding capacity (WHC), and flowability make it a useful ingredient in food products over chemical-based ones.

2. Polyglutamic Acid (PGA)

PGA is a naturally existing polymer with anionic properties. It is made up of a highly viscous homopolyamide comprising D- and L- glutamic acid units. PGA is produced by different microorganisms, but for commercial applications, Bacillus spp. (specifically Bacillus subtilis and Bacillus licheniformis) are generally employed [6]. Two distinct types of PGA exist α-PGA and γ-PGA. These forms differ in their structural composition, with glutamic acid components connected either by α-amino or γ-carboxylic group linkages. The linkages between the units in γ-PGA are predominantly γ-amide linkages, involving both γ-carboxylic acid and α-amino and units [16]. α-PGA is chemically synthesized; however, γ-PGA is primarily produced by a wide variety of microbial species, particularly Bacillus sp. unlike other proteins, it is not synthesized by ribosomes [17]. Instead, it is generated as an extracellular polymer by gram-positive bacteria [18] and a few gram-negative bacteria [19]. Bacillus subtilis and Bacillus licheniformis are the most common strains utilized for fermentative production of γ-PGA. However, B. anthracis, B. thuringensis, B. cereus, B. pumilis, B. megaterium, B. mojavensis, B. coagulans, Lysinibacillus sphaericus, Staphylococcus epidermidis, and Fusabacterium nucleatum were correspondingly reported for γ-PGA production [20,21,22,23,24,25]. In addition, a halophilic (salt-tolerant) archaebacterium called Natrailba aegyptiaca sp. is capable of producing γ-PGA. However, the challenges associated with its production make it unsuitable for fermentative cultivation of γ-PGA. γ-PGA exhibits the ability to stimulate and enhance immune activity, and it possesses functional properties for the targeted delivery of chemotherapeutic agents [26].

Proteins and peptides typically consist of α-amino and α-carboxylic acid units, which are prone to digestion by proteases. However, the presence of γ-amide linkages (involving α-amino and γ-carboxylic units) in γ-PGA prevents its degradation by proteases. γ-PGA is a chiral polymer with an optically active core in each glutamate residue. Three distinct types of stereochemically unique γ-PGA have been identified as homopolymer composed of D-glutamate units or L-glutamate units or copolymers containing both types. As γ-PGA in the culture medium, it forms a viscous solution containing approximately 5000 – 10,000 units of D- and L- glutamic acids [8]. The enzymes involved in the synthesis of γ-PGA have played a significant role in the development of production systems [27, 28]. The biosynthesis pathway of γ-PGA utilizes L-glutamic acid units, which can be obtained either externally or internally, with α-ketoglutaric acid serving as a direct precursor [2]. Due to its inherent differences from α-PGA (such as resistance to chemical modification and protease degradation), γ-PGA holds greater potential for medical applications, including vaccines, drug delivery, γ-PGA nanoparticles for localized drug release in cancer chemotherapy, and tissue engineering. The microbial synthesis of γ-PGA has fascinated significant consideration in the advancement of molecular biology techniques. Additionally, both isoforms of PGA have a broad range of applications, many of which are still being explored and discovered [28].

3. Biosynthesis of γ-PGA

A biosynthetic pathway has been proposed for the production of γ-PGA. The monomeric units of L-glutamic acid that combine to form γ-PGA can be acquired by two biosynthetic pathways i.e., exogenous or endogenous pathway (Figure 1). α-Ketoglutaric acid is a substrate for glutamic acid synthesis in the TCA cycle. For the endogenous production of γ-PGA, a shift in carbon source involving acetyl-CoA and TCA cycle intermediates is necessary. Glutamine synthase facilitates the conversion of exogenous L-glutamic acid into L-glutamine, which acts as a pioneer for glutamic acid synthesis. The synthesis of γ-PGA occurs via four distinct stages such as γ-PGA racemization, γ-PGA polymerization, γ-PGA regulation, and γ-PGA degradation. [29,30,31].

3.1. γ-PGA Racemization

γ-PGA is typically composed of different variants: D-glutamate, L-glutamate, or a grouping of both D- and L- enantiomers [32,33]. The process of incorporating D-glutamate into the growing L-glutamate chain is facilitated by a racemization reaction. This reaction converts L-glutamate, which can be obtained from external sources or produced internally, into D-glutamate. The conversion is carried out by the glutamate racemase gene (racE/glr), which plays a dynamic role in enabling the synthesis of γ-PGA. This enzymatic transformation allows the production of γ-PGA with a diverse composition of glutamate enantiomers [33].

3.2. γ-PGA Polymerization

Research studies have reported the existence of four genes (pgsB, C, A, and E) responsible for coding polyglutamate synthase (pgs), with ywsC, ywtAB, and capBCA identified as their Bacillus homologs [33,34]. Among these genes, PgsA plays a crucial role in removing the extended chain from the dynamic site, facilitating monomer expansion and the transportation of γ-PGA across the cell membrane. PgsB and PgsC are considered the key components of the catalytic site [28]. While PgsE is necessary for γ-PGA formation, high levels of pgsB, pgsC, and pgsA can contribute to the shaping of γ-PGA even in the non-availability of pgsE [34]. Additionally, the presence of Zn2+ is essential for γ-PGA production in B. subtilis, requiring the involvement of pgsE [35]. These studies demonstrated the intricate roles and interactions of multiple genes in the production of γ-PGA, highlighting the complex functions they perform collectively.

3.3. γ-PGA Regulation

The synthesis of γ-PGA involves the participation of two signal transduction systems: the ComP-ComA controller and the DegS-DegU, DegQ, and SwrA pathway [36]. The role of DegQ and its modification plays a crucial part in γ-PGA synthesis of γ-PGA and regulates the production of degradation enzymes [37]. It has been observed that the initiation of the pgs operon for γ-PGA synthesis occurs in the presence of SwrA and phosphorylated DegU (DegU-P), which triggers pgs expression when there are high levels of DegU-P, instead of swrA [38]. The transcriptional regulation of γ-PGA synthesis generally involves DegSU, DegQ, and ComPA, influenced by factors such as quorum sensing, phase variance signals, and osmolarity, while SwrA functions at a lesser extent [39].

3.4. γ-PGA Degradation

The degradation of γ-PGA is facilitated by two enzymes: Endo-γ-glutamyl peptidase and Exo-γ-glutamyl peptidase [4]. B. licheniformis and B. subtilis secrete Endo-γ-glutamyl peptidase, which breaks down high molar mass γ-PGA into smaller units ranging from 1000 Da to 20 kDa. This depolymerization process leads to a reduction in dispersity over time [40,41,18]. On the other hand, Exo-γ-glutamyl peptidase (Ggt), a vital enzyme in glutathione breakdown, is involved in the in vitro construction of γ-glutamic acid di- and tripeptides but is not directly involved in the in vivo synthesis of γ-PGA [33]. These enzymatic activities contribute to the degradation and metabolism of γ-PGA, influencing its molar mass and overall configuration.

4. Production of γ-PGA

Microbial production of γ-PGA is a promising approach for obtaining this biopolymer on a large scale. Various microorganisms, especially Bacillus species, have been studied and utilized for γ-PGA production. These microorganisms have the ability to synthesize γ-PGA through enzymatic pathways [10,34]. The production process involves the fermentation of selected microorganisms in a suitable growth medium. The growth medium composition is carefully designed to provide the necessary nutrients for the microbial cells to proliferate and produce γ-PGA. Typically, carbon and nitrogen sources, such as glucose, glycerol, or various amino acids, are included in the medium. In addition, other nutrients, vitamins, and minerals may be added to support cell growth and γ-PGA synthesis [10]. The fermentation process occurs in bioreactors under controlled conditions of temperature, pH, and oxygen supply. The microorganisms metabolize the nitrogen and carbon and sources in the growth medium, converting them into γ-PGA through enzymatic reactions. The synthesis of γ-PGA is regulated by specific genes and enzymes involved in its biosynthetic pathway [42]. During the fermentation process, the production of γ-PGA can be monitored by analyzing the culture samples at different time points. Techniques such as high-performance liquid chromatography (HPLC) or mass spectrometry (MS) can be used to quantify and characterize the γ-PGA produced. To optimize and improve γ-PGA production, various strategies can be employed. These include genetic engineering tactics to enhance the expression of genes involved in γ-PGA synthesis, optimization of fermentation conditions, and the use of advanced bioprocess technologies [10].

Four different approaches are employed for the production of γ-PGA: microbial fermentation, peptide synthesis, chemical synthesis, and biotransformation [43]. γ-PGA is a component commonly found in Japanese traditional food, specifically natto, which is prepared from fermented soybeans using Bacillus strains [44]. Research has been done to study the nutritional requirements for γ-PGA production in order to improve its fermentation productivity. Bacillus strains are proficient of making a huge amount of γ-PGA, up to 50 g, in the growth medium. Based on their nutritional requirements, bacterial species can be classified into two categories: one relies on L-glutamic acid as the nitrogen and carbon source for growth and γ-PGA production, while the other does not depend on L-glutamic acid. In either case, a sufficient amount of nitrogen and carbon and sources, typically in the range of 2-20 grams, is required by the bacteria for γ-PGA production. Different bacteria employ distinct γ-PGA production systems, indicating variations in the synthesis process [45]. Bacillus subtilis IFO3335, for instance, can synthesize a substantial amount of 0.96 g γ-PGA in a 100 mL medium containing 3 g of L-glutamic acid, 2 g of citric acid, and 0.5 g of ammonium sulfate, without any polysaccharide by-products [46]. When no or only 0.5 g of L-glutamic acid is added to the same medium with 2 g of citric acid and 0.5 g of ammonium sulfate, a small amount of PGA is produced. Interestingly, the addition of 0.01 g of L-glutamine to the medium leads to a significant increase in γ-PGA production, while the utilization of 0.1 g of yeast extract or glucose results in little to no γ-PGA production. Thus, the combination of citric acid and ammonium sulfate in the cultivation medium, supplemented with small amounts of specific components like L-glutamine, promotes efficient γ-PGA synthesis [47]. γ-PGA production has been also achieved through co-expression in E. coli. The γ-PGA synthase genes pgsBCA and racE from an L-glutamate-dependent γ-PGA producer B. licheniformis NK-03 and a non-L-glutamate-dependent γ-PGA producer B. amyloliquefaciensLL3 were cloned and co-expressed in E.coli JM 109 for evaluation of γ-PGA productivity. Results showed that pgsB and pgsC of both strains are highly similar with 93.13 and 93.96% resemblance, where the pgsA and racE presented 78.53 and 84.5% similarity, respectively [48].

5. γ-PGA from Bacillus spp.

Extensive research has been performed on the production and applications of γ-PGA (Table 1). PGA produced chemically results into the production of low molecular mass i.e., 10 kDa limiting its application. However, the γ-PGA (bacterial) varies from 10-100 kDa and may often reach as high as 10000 kDa [49]. Several micro-organisms are involved in the synthesis of γ-PGA. Bacillus sp. like B. subtilis and B. licheniformis are used to produce it commercially. γ-PGA production from B. subtilis (natto) proved that synthesis or lengthening of γ-PGA is coupled with its degradation and the resultant γ-PGA synthase complex is unstable. However, it has been found that B. subtilis (chungkookjang) produce an ultra-high molecular mass of γ-PGA in a medium containing a high concentration of ammonium sulphate. Without any by-products, the average high-molecular mass of γ-PGA obtained is 2×10⁶. γ-PGA with a molecular mass exceeding 2 × 10⁶ Da was challenging to measure accurately, and high-molecular-mass γ-PGA was estimated to be approximately 7 × 10⁶ Da [50,51]. There are two types of microorganisms involved in the production of γ-PGA, namely L-glutamic acid-dependent and -independent bacteria [34]. L-glutamic acid-dependent bacteria include B. subtilis (chungkookjang) [52], B. subtilis (natto) ATCC 15245 [53], B. subtilis CGMCC 0833 [54], B. licheniformis NK-03 [55], and B. licheniformis 9945a [56]. On the other hand, non-glutamic-acid-dependent bacteria include B. amyloliquefaciens LL3 [22], B. subtilis C1 [57], and B. subtilis C10 [58].

The bacterium Bacillus subtilis NRRL-B2612, when 200 g/L wheat gluten is used as a constituent and cultured at 33 ℃ for 2-3 days 10-14 g/L γ-PGA was produced [19]. Bacillus licheniformis ATCC 9945A was reported to produce 23 g/L of the product in the medium consisting 20g/L of glutamic acid,80g/L of glycerol, 12 g/L of citric acid, 7 g/L of ammonium chloride when cultivated at 37 ℃ for 4 days [59]. The strain Bacillus subtilis F-2-0, medium constitutes glutamic acid 70 g/L, glucose 1 g/L, veal infusion broth 20 g/L. 45.5 g of γ-PGA was produced when cultivated at 37 ℃ for 2-3 days [60]. Another bacterium employed was Bacillus licheniformis and the medium was LB agar slant composed of yeast extract 5 g/L, tryptone 10 g/L, NaCl 10 g/L, agar 15 g/L. Batch fermentation experiments were carried out in 250 mL Erlenmeyer flasks consisting of 50 mL of the sterile cultivation medium at 37±2 ℃ and 200 rpm for 72 h. The production medium was inoculated with 2% of 12 h inoculum and kept for fermentation. This was followed by centrifugation at 12000 rpm for 20 min at 4 ℃ to separate cells from the cultured broth. The cell-free supernatant containing γ-PGA was kept at 4 ℃ overnight after pouring it into 4 volumes of ice-cold ethanol with gentle stirring. The γ-PGA precipitate was collected by centrifugation at 12000 rpm for 20 min 4 ℃. The crude γ-PGA was resuspended in deionized water and any insoluble particles were pelleted by centrifugation at 12000 rpm at 4 ℃ for 20 min for its removal. The precipitation steps were followed thrice, and the resultant γ-PGA was centrifuged again and dried at 70 ℃ until it attained a constant weight [61]. Further purification was done by dialysis and lastly, γ-PGA slurry was freeze-dried to prepare powder and was estimated by ninhydrin with glutamic acid as a standard by TLC [62].

5.1. B. licheniformis

B. licheniformis, particularly the strain B. licheniformis 9945a (NCIM 2324), is a well-known and extensively utilized bacterium for the production of γ-PGA. To achieve maximum yield, the production was optimized through solid-state fermentation. The impact of various factors such as substrates, carbon and nitrogen sources, moisture content, pH, amino acids, and TCA cycle intermediates on γ-PGA production was investigated using the "one factor at a time" approach. By employing optimized media, a yield of 98.64 mg (g dry solids)-1 γ-PGA was obtained through solid fermentation [63]. Bajaj et al. (2009) [64] also conducted research on optimizing the production of γ-PGA using B. licheniformis NCIM 2324, employing the "one factor at a time" method. They utilized response surface methodology to determine the optimal nutrient concentrations, which were then experimentally validated. The optimized medium, consisting of glycerol (62.4 g l^-1), citric acid (15.2 g l^-1), ammonium sulfate (8.0 g l^-1), and L-glutamic acid (20 g l^-1), resulted in a yield of 26.12 g l^-1 of γ-PGA. In comparison, the yield obtained with the basal medium was 5.27 g l^-1. The γ-PGA produced had a molecular mass of approximately 2.1 x 10⁵ Da.

B. licheniformis Al3, a producer independent of exogenous glutamate, achieved a γ-PGA yield of 28.2 g l^-1 in an optimized medium. The optimized medium consisted of glucose (50 g l^-1), NH₄Cl (3 g l^-1), yeast extract (2 g l^-1), MgSO₄.7H₂O (0.8 g l-1), NaCl (0.8 g l^-1), CaCl₂.2H₂O (0.00084 g l^-1), K₂HPO₄ (6.4 g l^-1), FeSO₄.4H₂O (0.006 g l^-1), 0.1 mL of trace element solution, and a culture volume of 23 mL. The Plackett-Burmann design was used up to 72 hours after inoculation to assess the effects of different factors on γ-PGA production [65]. The results indicated that yeast extract and medium volume were the two factors that significantly influenced γ-PGA production. For the bacteria B. licheniformis WBL-3, monthly subculture was performed on agar slants containing 2.0% agar. The slants consisted of 10 g citric acid, 10 g L-glutamic acid, 6 g NH₄Cl, 1 g K₂HPO₄, 0.05 g MgSO₄.7H₂O, 0.02 g FeCl₃.6H₂O, 0.2 g CaCl₂, and 0.05 g MnSO₄.H₂O at pH 6.5 [66]. The same medium without agar was used for seed medium (50 mL) preparation and incubated at 37 ℃ for 24 hours. The flasks were placed in a rotary shaker at 200 rpm [67]. In the case of B. licheniformis A35, under denitrifying conditions, it produced 8 mg/mL of γ-PGA. The pre-cultured medium used in a liter of culture contained 10 g meat extract, 10 g peptone, 5 g sodium chloride, and 10 g glucose [68].

5.2. Bacillus subtilis

Bovarnick (1942) was the first to demonstrate that B. subtilis fermentation released the γ-PGA into the medium [69]. More emphasis has been placed on investigating B. subtilis strains for γ-PGA production compared to B. licheniformis. Scoffone et al. (2013) evaluated γ-PGA production by knocking out the pgdS and ggt genes, which are responsible for two important γ-PGA degradation enzymes, in the laboratory strain B. subtilis 168. The impact of single mutations (deletion of pgdS or ggt) and a double mutation (deletion of both pgdS and ggt) on γ-PGA production was assessed. While single mutations did not result in significant improvement in γ-PGA yield, the double mutant strain produced more than twice the amount (>40 g L-1) compared to the wild-type strain [70]. Shih et al. [71] presented findings on the high-yield, cost-effective, and large-scale production of γ-PGA from B. subtilis ZJU-7 (B. subtilis CGMCCl250). Their study demonstrated that using 40 g/L yeast extract, 30 g/L L-glutamate, and 20 g/L initial glucose, along with maintaining a glucose concentration in the range of 3-10 g/L through a fed-batch approach, significantly improved the yield of γ-PGA. Compared to batch fermentation, this approach resulted in a 1.4 to 3.2-fold increase in γ-PGA yield. The study recorded an overall γ-PGA concentration of 101.1 g/L and a productivity of 2.19 g/L. The strain Bacillus subtilis ZJU-7 is obtained from fermented bean curd. The culture medium used for slant preparation consists of glucose (10 g/L), tryptone (10 g/L), L-glutamic acid (10 g/L), and NaCl (5 g/L). The seed medium is composed of the same components as the slant medium, with the addition of 0.1 g/L MgSO₄ and 0.1 g/L CaCl₂. The basal medium is similar to the slant medium but contains a higher concentration of L-glutamic acid (20 g/L). To optimize the effects of these components on γ-PGA production, response surface methodology (RSM) is employed by changing the composition of the media. The pH of the media is adjusted to 7.0 using HCl or NaOH, and all media samples are sterilized by autoclaving at 121 ℃ for 20 minutes. For cultivation, the inoculated samples are transferred into 500 mL flasks and incubated at 37 ℃ with shaking at 200 rpm. After a fermentation period of 24 h, the culture is separated, and the γ-PGA is purified through methanol precipitation [46].

Batch cultures of Bacillus subtilis (natto) were conducted in a 5L laboratory fermenter system, while a 600-liter pilot plant fermenter system was employed for the development process. Agar plates for culturing were prepared using a 1.5% agar solution. The medium used consisted of 8% glucose, 10% sodium L-glutamate, 0.05% K₂HPO₄, 1.5% peptone, 0.02% CaCl₂, 50% biotin, 1.0% yeast extract, and 3.0% NaCl. Additionally, 0.05% silicone oil (Dow Corning Silicone) was included as an anti-foaming agent, and the temperature was maintained at 37 °C. The extracellular production of γ-PGA was observed with a molecular weight ranging from 100,000 to 2,500,000 Da. Alcohol was used to precipitate γ-PGA from the cell-free culture broth solution, followed by centrifugation and purification of the precipitates [67].

For Bacillus subtilis strain MR-141, derived from strain MR-1, spore formation was achieved by growing the strain on nutrient plates containing 1.5% agar at 40 °C for 7 days. Subsequently, the strain was transferred to MSG medium, which consisted of 6% maltose, 7% soy sauce, 3% sodium L-glutamate, 0.25% K₂HPO_4, 0.05% MgSO₄.7H₂O, and 3% NaCl. Alternatively, MSG medium with 6% glucose instead of maltose could be used, along with 0.1% silicone oil as an anti-foaming agent. The glutamic acid present in the broth was quantified using an amino acid analyzer [72].

5.3. Bacillus anthracis

B. anthracis, a known producer of enantiomer form of γ-PGA does not release γ-PGA into the medium as compared to other Bacillus species instead, it is peptidoglycan bound [73]. It is important to note that industrial production of γ-PGA by B. anthracis is not viable owing to its toxicity. γ-PGA aids in making the B. anthracis capsule non-immunogenic, which has been linked to the lethal toxin [74]. Hence, its cap gene responsible for the anchoring of γ-PGA onto its surface needs to be targeted to render B. anthracis immunogenic [27].

5.4. Bacillus thuringiensis

B. thuringiensis sv. monterrey strain BGSC 4AJ1 and B. anthracis (Ames) have four common alleles, gmk-1, pta-1, pur-1 and tpi-1, where other three alleles, glpF-57, ivld-52, and pycA-52, differ by 2, 2 and 3 nt respectively. Genes encoding the synthesis of γ-D-PGA showed similarity with those of B. anthracis and are present on a plasmid (pAJ1-1). The Discovery of a γ-PGA capsule in this B. thuringiensis strain is an indication of the ability of the bacteria to be pathogenic under certain conditions [7].

6. Structural and Physico-Chemical Properties of γ-PGA

γ-Polyglutamic acid (γ-PGA) exhibits diverse properties, including various conformational states, enantiomeric forms, and molecular weights. Its biodegradable, non-toxic, and non-immunogenic characteristics make it valuable in the food and pharmaceutical industries. For instance, Zhang et al. demonstrated that utilizing waste biomass hydrolysate and substituting tryptone in the γ-PGA production medium introduces a more sustainable production method. Applications of γ-PGA encompass protein crystallization, tissue adhesives for soft tissues, and non-viral vectors for gene delivery. Each unique property of γ-PGA aligns with specific applications, highlighting the need for further research to identify bacterial strains capable of producing high yields of γ-PGA with tailored properties [75]. Optimization of γ-PGA production concerning its production cost, molecular mass, and conformational or enantiomeric properties is crucial in bringing its application into practice. Knowledge of the enzymes and genes involved in γ-PGA production will not only aid in increasing productivity by reducing the production cost but also help in understanding the mechanism by which γ-PGA is beneficial in numerous applications [7]. Physicochemical and functional characterization of γ-PGA molecules can be achieved using several modern techniques and instruments (Figure 2).

6.1. Fourier Transform Infrared (FT-IR) Spectroscopy

FT-IR spectroscopy is the measurement of the procedure applied to record IR spectra. FTIR interferograms expose the functional groups in the purified γ-PGA illustrates that can be resolved by recognizing the specific peak values in the graphical values of FTIR. FTIR spectroscopy was carried out for the detection of γ-PGA functional groups of 4000-400 cm^-1 frequency. The sample pellet for the spectrum analysis was prepared using purified γ-PGA and dried potassium bromide (KBr) by compression and the functional group vibrations for C=O (carboxyl), -NH (amine), -OH (hydroxyl), and C-N (carbonyl) stretches were produced as various peaks and bendings [67,77].

6.2. Nuclear Magnetic Resonance (NMR) Analysis

Usually for spectroscopy ¹H- and ¹³C-NMR are used to analyze the degree of esterification and homogeneity of γ-PGA. Following NMR spectra chemical shifts are standardized using the known standards. The samples were analyzed at 100 MHz, with a 30°pulse and a 4s cycle time. As well, solid state samples were analyzed at 50 MHz, and the spectra were kept under careful observation of cross-polarisation, magic angle sample spinning, and power decoupling circumstances with a 90° pulse and 4 s cycle time. To know the chemical composition comprehensively, analyses are done. ¹H NMR for γ-PGA in D₂O shows chemical shifts at 3.66 ppm for the α-CH proton, 2.08 ppm for the ꞵ-CH₂ proton, and 2.33 ppm for γ-CH₂ proton. The ¹³C NMR spectra showed chemical shifts at 55.53 ppm for α-CH₂ group, 27.77 ppm for ꞵ-CH₂ group, 34.34 ppm for γ-CH₂ group, 175.21 ppm for the CO group, and 181.96 ppm for the COO^- group [67].

6.3. Thermal Analysis

In thermogravimetric analysis (TGA) of γ-PGA, thermal degradation temperature and thermal stability of biomolecules can be determined by using a thermal gravimetric analyzer. In dynamic experiments, usually, a powdered form of purified γ-PGA was used. The TGA was implemented at 50-700 ℃ temperature and a heating rate of 10 ℃ min^-1 at a nitrogen atmospheric rate of 25 mL min^-1 [78]. TGA is carried to determine the thermal decomposition temperature (T_d). It expresses the thermal stability of γ-PGA and gives the thermal gravimetric curve and the percentage loss in weight was analyzed while raising the temperature from 50 ℃ to 700 ℃. γ-PGA produced in SJ-10 was found to exhibit high resistance to high temperatures of thermal degradation. The decomposition temperature (T_d) and the temperature representing half of the initial weight (T_d50%) were recorded at 320 ℃ and 455 ℃, respectively. The T_d was the same as the sodium form of γ-PGA when compared to the normal one. The T_d of γ-PGA was different according to the combined cations. The T_d of γ-PGA in addition to Na^+, K⁺ Ca²⁺, and Mg²⁺ cations was more than those with H⁺ and NH⁺₄ [67,79].

6.4. Molecular Weight Determination

The γ-PGA synthesized by Bacillus spp. typically exhibits a high molecular weight ranging from 10⁵ to 10⁶ Da [34]. Molecular weight estimation of γ-PGA has predominantly been conducted using gel permeation chromatography (GPC), employing various mobile phases and calibration against different standards [56, 80]. Bajestani et al. reported utilizing DEAE cellulose-52 column resin to run ion-exchange chromatography. The column charged with γ-PGA was eluted with a gradient concentration of NaCl (0.1, 0.5, 0.75, and 1 M), and fractions were collected. Then, γ-PGA content was quantified using (GPC) as it detects UV-light roughly at 216 nm depicting the chromatogram, followed by lyophilization. The molecular weight of γ-PGA as a heavy weight fraction was estimated to be 7.7x10⁶ g/mol and 1.7x10⁴ g/mol as the average molecular weight number. Birrer et al. followed another approach of chromatography, high-performance liquid chromatography (HPLC), and GPC to determine the number (M_n) and weight average molecular weights (M_w) along with polydispersity of γ-PGA. A calibration curve was constructed using narrow polydispersity pullulan standards and molecular weight M (M_w and M_n) of γ-PGA was calculated to be 22000 g/mol and 266000 g/mol, respectively [56].

6.5. Amino Acids and Enantiomeric Composition Analysis

HPLC is usually used to serve this purpose. The γ-PGA hydrolysate chromatogram was detected at a position corresponding to D-glutamic acid having equal retention flow, no peak corresponding to L-glutamic acid was detected. The result indicates that separated biocompatible γ-PGA contains D-glutamic acid residues the most [7].

7. Physico-Functional Properties

7.1. Water Holding Capacity

A substance capacity to retain moisture is water holding capacity. γ-PGA is reported to have an excellent water-holding ability [82]. Apart from food applications, γ-PGA is used in cosmetic industries because of its significant water-holding capacity, and hydrogels are utilized for biomedical applications [83]. Additionally, the introduction of γ-PGA to sandy soils has been reported a significant lowering of the water insinuation competence, whereas the water holding capacity of the soil improved the saturated water content and effective water utilization. The results in soil suggest that γ-PGA can not only add to the water-holding capacity of soil but bring about an obvious change in the moisture distribution patterns thus, paving a way through agro-ecosystems as well [84]. The good water-binding capacity of γ-PGA results in an increase in moisture holding while reducing the oil uptake significantly [85]. However, the water holding capacity of γ-PGA was found to be dropped (56.9%) when the reaction time was increased up to 9 days [86].

7.2. Emulsifying Property

The study on the effect of γ-PGA addition on the emulsifying property of sponge cake revealed that the addition of γ-PGA significantly improved the emulsion activity and stability, and foam stability of sponge cake paste confirming the contribution of γ-PGA in delayed staling by [82]. A multiple layered oil-in-water emulsions of γ-PGA with soyabean oil showed that the emulsion ability was sturdily reliant on γ-PGA addition. A sheer increase in mean particle diameter was detected with a surge in γ-PGA concentration (0 to 0.01 w/v %) and an appreciable cream formation occurred at intermediate γ-PGA concentrations (0.023 w/v %) [89].

7.3. Rheology and Viscosity

The rheology studied by Zhang et al. focused on the rheological properties of γ-PGA produced by Bacillus subtilis 1006-3. The γ-PGA solution exhibited non-Newtonian fluid behavior, specifically pseudoplasticity, with shear-thinning properties. This behavior is described using the Ostwald-de Waele power law model. The apparent viscosity of the γ-PGA solution increased as its concentration was raised from 1-10%. Deviations from a neutral pH, as well as the addition of NaCl or MgCl₂, reduced the apparent viscosity of the γ-PGA solution. The solution was more sensitive to the addition of Mg²⁺ ions compared to Na⁺ ions. At concentrations of 4, 6, and 8%, the γ-PGA solution showed a predominantly viscous response (G”>G’) within the angular frequency range of 0.1-100 rad/s. The study indicated the potential application of γ-PGA as a thickening agent due to its rheological properties [88].

8. Biological Properties

8.1. Antioxidant Activity

The ABTS radical scavenging and phosphomolybdenum assay was accomplished to measure the total antioxidant activity of γ-PGA. The γ-PGA from Bacillus sp. SJ-10 with a molecular mass of 400 kDa unveiled a maximal scavenging activity at 1 mg/mL (20 µg ascorbic acid-equivalent). Additionally, it exhibits action on par with commercially available natural antioxidants [86]. The anti-oxidant activity of γ-PGA paves its way into various fields of food, cosmetic and biomedical industries.

8.2. Anti-Microbial Activity

The γ-PGA was reported to have an inhibition effect towards both Gram-positive and Gram-negative bacteria. S. aureus was inhibited strongly by the γ-PGA. γ-PGA exhibited an anti-bacterial activity against these bacteria at 1% concentration but showed no activity at 0.1%. Apart from that, the γ-PGA showed no activity against pathogenic yeasts, C. albicans [86]. Due to its excellent anti-microbial activity, γ-PGA is used as hydrogels of a new wound dressing type for wound healing also, superior effects in healing were observed when compared to sutures, component skin adhesives, and fibrin glue with reduced inflammatory response [90].

8.3. Angiotensin-Converting Enzyme (ACE) Inhibitory Activity

γ-PGA is reported to have significant ACE-Inhibitory activity, ACE inhibition regulates blood pressure by getting involved in the renin-angiotensin system therefore, is an important pathway in treating hypertension in humans. ACE-inhibitory activity in γ-PGA possibly due to oligosaccharides and protease inhibitors connected with hydroxyl groups to form hydrogen bonds with ACE, besides peptides that may be present in γ-PGA [91]. γ-PGA showed an increase in ACE inhibition in a concentration dependent manner. The highest ACE- inhibition activity was 100% observed from 1-1.8 mg/mL γ-PGA which endured persistent thenceforth [91]. The Inhibitory concentration (IC₅₀) value of γ-PGA was found to be 0.108 mg/mL which is lower than the standard ACE inhibitory drug named captopril (IC₅₀) 0.247 mg/mL [86].

8.4. Wound Healing

γ-PGA with the other electrolytic materials like chitosan formed a polyelectrolyte composite and proved to be a potential wound dressing material by regulating the water uptake and thus minimizing the risk of dehydration on the wound. It also improved the structural stability of chitosan [92]. Also, γ-PGA-PEG (Polyethylene glycol) injectable complex was shown to be a promising hemostatic material for liver and spleen injuries (visceral hemorrhage) when compared to clinical fibrin glue [93].

9. Applications of γ-PGA

Poly-γ-glutamic acid (γ-PGA) is a biodegradable, water-soluble, and non-toxic biopolymer produced by various Bacillus species. Its unique properties make it suitable for diverse applications in food, pharmaceuticals, agriculture, and water treatment (Figure 3). Also, various commercial PGA have been studied for their functional properties and further applied in diverse applications (Table 2).

9.1. Flocculation

γ-PGA can be used as a bio-flocculent in wastewater treatment, downstream processing in food industries, and pharmaceutical and medicine industries. γ-PGA is used for the flocculation of solid waste and metals in wastewater treatments [94]. The flocculation efficiency has a direct relation with the molecular mass. γ-PGA from B. subtilis P-104 was shown to have good flocculating activity [95]. It can be improved by the addition of cations by stimulating the flocculation activity by nullifying and alleviating the negative charge on the functional group of bio-flocculent by establishing bridges amid elements. Cations, temperature, and pH are the major factors that affect the flocculation efficiency of γ-PGA. A new organic approach for solving consequential environmental issues generated by the use of massive quantities of liquid fertilizer in agriculture: limiting surplus ammonia in soil and thus nitrogen translation into γ-PGA has been reported. For cations like Fe²⁺, Fe³⁺, Mg²⁺, Ca^2+, and Mn²⁺, that occur naturally, γ-PGA serves as a waste nitrogen transit base as well as an environmentally safe fertilizer/manure [96]. γ-PGA (9.9 x 10⁵ Da) could be used for the elimination of basic dyes from aqueous solution. The progression is a result of the electrostatic interface of γ-PGA and dyes which initiates adsorption at pH >5 and the exclusion of dyes from γ-PGA takes place at extremely acidic (pH 1), facilitating the reuse of γ-PGA [97]. In addition to its benefit as a flocculating agent, PGA when used as an inorganic salt may result into the production of raw sludge that has to be managed later [98].

γ-PGA is a flocculant as it can play an important role in effluent treatment, and downstream processing in foods, pharmaceuticals, and drug industries therefore can replace synthetic flocculants. γ-PGA can be used as a bio-flocculant in food and fermentation industries to harvest microalgae. Reaping microalgae with PGA is lucrative, and during other harvesting techniques like centrifugation, loss of lipid is prevented due to algal cell breakage [99]. γ-PGA from B. licheniformis CCRC 12826 revealed efficient flocculation of numerous organic and inorganic compounds [45].

9.2. Bioremediation

Pollutants in the environment like heavy metals, radionuclides, and synthetic substances, endanger public health and upsurge universal lack of provisions because of contamination which leads to polluted water, diminished agricultural output, and adverse effects like acid rain. The remediation of polluted soils, residues, and streams includes the interaction of these contaminations with γ-PGA to implement new remediation strategies [100].

Removal of heavy metals: γ-PGA covalently combined into microfiltration layers through membrane pore surface attachment has a super-high heavy metal sorption capability. γ-PGA muddles and effectively eliminates >99.8% of lead ions from water using a low-pressure ultra-filtration system [101].

Dye removal: γ-PGA (9.9×10⁵ Da) is an efficient method for removing simple dyes from hydrated solutions. At pH 1, it was found that 98% of the adsorbed dye on γ-PGA might be retrieved, allowing γ-PGA to be reused [97].

9.3. Fertilizer

Plant growth and development are enhanced by adding fertilizers to the soil. To avoid environmental pollution, γ-PGA can be used as a bio-control agent and/or a synergist to chemical fertilizers in agriculture. It assists in enhancing growth by improving nutrient consumption even in exhausted nutrient situations. The enzymes from soil such as urease, sucrose, and catalase show an augmented activity after the supplementation of γ-PGA, nitrogen-immobilized microbes raise the total nitrogen accretion in soil [102]. γ-PGA was found to promote the growth of Chinese cabbage and increase the total nitrogen, soluble protein, and soluble amino acids content in leaves. The addition of γ-PGA brings upon a surge in the activity of enzymes involved in the breakdown and acclimatization of nitrogen. It facilitates the Ca influx in the cytoplasm which acts as a positive signal for nitrogen metabolism thus promoting the growth of plants [102]. Wang et al. (2024) reported the use of fermented grain broth BSG which is a good source of live B. subtilis and other metabolites beneficial to soil and plant, thus tends to be used as a modern functional bioorganic fertilizer [103]. B. subtilis B6-1 produces Lipopeptides and γ-PGA using soybean and sweet potato scums sufficiently repressed cucumber wilts, amplified the growth of cucumber seedlings and also increases nutrient consumption [104].

9.4. Cryoprotectant

γ-PGA possesses a high anionic amino acid composition and thus exhibits an antifreeze activity. Polymers having acidic amino acids possess high antifreeze activity in comparison to other polymers. γ-PGA with lower molecular masses <20kDa, demonstrated significant antifreeze activities than very effective antifreeze agents like glucose, without interfering with the taste of foods. The antifreeze activity is reduced in the sequence Na salt = K salt > Ca salt > acidic form [105]. During freeze drying, γ-PGA from B. subtilis has the potential to shield probiotic bacteria Lactobacillus paracasei remarkably better than sucrose. The probiotic strains of Bifidobacteria (Bifidobacteria longum and Bifidobacteria breve) have been in use for proper operation of the gastrointestinal tract. γ-PGA helps protect these cells in fruit juices and prevents their survival from harsh environments of the digestive tract [106].

9.5. In Food and Medicine

γ-PGA is utilized as a food constituent due to its functional and physico-chemical features. Consumption of γ-PGA improves intestinal calcium absorption in post-menopausal women by inhibiting the formation of an insoluble calcium complex with phosphate and can potentially be used for the treatment of bone disorders [107]. Supplementation of γ-PGA acts as a preventing agent for osteoporosis of bones by greatly improving in vitro and in vivo calcium solubility in rats and postmenopausal women respectively, also the calcium content of their bones [107, 7]. γ-PGA conjugates to produce increased absorption of vanadyl sulphate which is a mimetic insulin inorganic salt. γ-PGA has an anti-diabetic effect because it reduces the rate of intestinal absorption of glucose, as γ-PGA vanadyl complex having higher insulin – mimetic activity than free vanadyl sulfate. K-γ-PGA administration prevents a surge in blood pressure by tumbling sodium absorption and thus controls hypertension [108]. γ-PGA was found to improve the gut microbiota by increasing the abundance of Lactobacillales in the gut [109].

γ-PGA has a significant antifreeze activity that’s why it acts as a cryoprotectant for frozen foods. As a cryoprotectant, during freeze-drying, the impact of γ-PGA probiotic microbes was found to be more effective than sucrose, sorbitol, and trehalose [110], and Acetobacter xylinum produces nata, bacterial cellulose [111]. Also, it is used as a thickening agent in foods/beverages improves the texture of foods, and also prevents aging. γ-PGA is demonstrated to have a positive effect in dropping oil uptake and moisture loss during deep fat frying of foods. γ-PGA is known to have water retention capacity therefore, it helps in controlling water loss and produces a dense matrix with improved integrity. Thus, γ-PGA can be utilized as a functional oil-reducing agent in deep-fat fried foods. During deep-fat frying, the impact of γ-PGA on the absorption of oil and loss of moisture content in doughnuts was found to be more effective and has preferred appearance and taste over ordinary doughnuts. Subsequently, in deep-fat fried foods, γ-PGA can be utilized as a functional oil-reductant [85].

9.6. Cosmetics

γ-PGA plays a significant role in cosmetics because γ-PGA improves the solubility of vitamin C when forms the PGA– vitamin C complex. Vitamin C is crucial for collagen creation which assists in skin repair. Due to its anti-oxidant activity, it acts as anti-aging. Therefore, it is a dynamic component in cosmetic compositions owning to its hygroscopic properties and skin compatibility. γ-PGA is a good hydrophilic humectant and has the potential to improve the production of urocanic acid, pyrrolidone carboxylic acid, and lactic acid compared to hyaluronic acid and soluble collagen as natural moisturizing agents. γ-PGA aids in enhancing the qualities of skincare and hair care products, such as exfoliating, nourishing, and taking away wrinkles [112]. The cosmetic constituent with γ-PGA-vitamin complex results in better firmness, enhanced absorption, and constant release of vitamins from the composite [11].

9.7. Biomedical Applications

γ-PGA gained its space in biomedical applications due to its glutamic acid composition which are natural excerpts of the human body [113].

9.7.1. Hydrogels

Hydrogel is a bioabsorbable product known to have the ability to swell in water and retain it inside its structure. It has paved the way for immense applications in the field of drug delivery and tissue engineering. Hydrogel preparation has various approaches, including γ-irradiation, chemical, or physical cross-linking. Microbial γ-PGA and L-lysine were cross-linked to prepare biodegradable hydrogels by amide bond in the presence of DMT-MM in water [114]. Using no chemical treatment γ-PGA reacted with polyvinyl alcohol (PVA) in aqueous solution to form hydrogel. The elongation and water retention ability of the hydrogels is increased with an increase in γ-PGA concentration. Protein adsorption and platelet adhesion on hydrogel have an inverse relation with γ-PGA concentration and thus help in improving the blood compatibility of the hydrogel. Due to its water resistance, mechanical properties, and blood compatibility, PGA–PVA hydrogel functions as a good biomaterial for medical devices that are used to carry blood [115]. The combination formed with bacterial cellulose and γ-PGA was found to have promising applications as bio-degradable structural high-performance materials, construction materials, and tissue engineering scaffolds (tendon, ligament, and skin) due to their biodegradability and good tensile toughness [116]. γ-PGA hydrogel showed a promising result as an edible coating material in shiitake mushrooms preserving its nutrient quality and extending the shelf-life [117].

9.7.2. Nanoparticles

Gene and Drug delivery could be made possible by nanoparticles. Due to the smaller size of the nanoparticle, it can easily escape from the reticuloendothelial system resulting in increased circulation time in blood. γ-PGA being hydrophilic and water soluble is used as a carrier for anti-cancer drugs. The γ-PGA and chitosan nanoparticles have been widely used for the oral conveyance of hydrophobic drugs and proteins. PGA-chitosan nanoparticles act as an efficient system for the delivery of insulin to diabetic patients for treatment [115,118,119].

9.7.3. Tissue Engineering

Tissue engineering is the process by which biological substitutes are developed to reinstate and sustain the functions of tissue. Due to its hydrophilic and cytocompatible nature γ-PGA/chitosan composite biomaterial exhibits latent application in tissue engineering than traditional chitosan matrices [120]. A PEC (poly electrolyte complex) of chitosan and γ-PGA shows a potential application in wound dressing. The complex holds the required moisture and has good mechanical properties, which allows easy removal of the dressing from the surface of the wound without destroying the renewed tissues [92].

9.7.4. Drug Carrier/Deliverer

As a drug delivery agent, the factor determining the drug delivery properties involves the molecular mass of γ-PGA, which helps to regulate the rate of drug discharge. γ-PGA with covalently attached cisplatin reduces cisplatin toxicity, improves tumor size retention in naked mice with xenografted human breast tumors, and extends the survival of bare mice with Bcap-37 tumor cells [121.

9.7.5. Metal Chelators

Heavy metals and radionuclides may be removed using metal chelators. PGA-coated super paramagnetic iron oxide NPs had a high efficiency in removing heavy metal from activated gastrointestinal fluid and a metal solution. [77]. For instance, γ-PGA molecular mass or sub-atomic mass of ~3-6 x 10⁴ Da was utilized to deliver Paclitaxel poliglumex (a macromolecular form of paclitaxel and γ-l-PGA) exhibits advantages over ordinary paclitaxel. The active agent paclitaxel was eventually released from paclitaxel poliglumex as it accumulated in tumor tissue [123].

9.7.6. Biological Adhesive

A hydrogel formed by a mixture of γ-PGA aqueous solution and gelatin in the presence of water-soluble carbodiimide helps in lung adhesion and air-leak sealing than regular fibrin glue [124].

10. Conclusions

γ-PGA is a biopolymer that is edible, non-immunogenic, and therefore can be used without any worry in numerous different applications that are expanding rapidly. γ-PGA is an important polymer due to its various applications in the fields of medicine, agriculture, wastewater treatment, and food industries. In particular, γ-PGA is an enormously promising constituent in food. As a biodegradable substance, γ-PGA possesses a few preponderant features containing good water solubility, biocompatibility, degradability, and non-toxicity. Based on this, γ-PGA can be used in pharmaceuticals, such as drug carriers/deliverers, vaccine adjuvants, and coating material for microencapsulation, etc. PGA as humectants regulate water activity in foods, improving stability and viscosity, maintaining texture, and reducing microbial activity. Food additives help to reduce water activity while keeping foods moist and safe for a longer shelf life of processed foods. Also, many researchers have conducted extensive research on the cost-effective manufacture of γ-PGA.