γ- PGA is a naturally occurring biopolymer synthesized by various microorganisms, particularly species of Bacillus. The report delves into the challenges and advancements in cost-effective production strategies, addressing the economic constraints associated with large-scale γ-PGA synthesis. Its biocompatibility, biodegradability, and non-toxic nature make it a promising candidate for diverse industrial applications. γ-PGA’s exceptional water-holding capacity and humectant properties are key to its utility in the food industry. These features enable it to enhance the stability, viscosity, and shelf life of food products, making it a valuable ingredient in processed foods.
Introduction
Biopolymers, defined as naturally occurring 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., polypeptides, polysaccharides, and polynucleotides, based on their monomeric units, possess remarkable versatility and can be easily manipulated and modified to suit a wide range of applications.
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 and the enhancement of antioxidant and anti-inflammatory properties. Depending on the fermentation conditions and food substrate, various bioactive metabolites, including biopolymers such as polyhydroxyalkanoates, polylactic acid, and polyglutamic acid (PGA) are produced by non-infectious Bacillus and Lactobacillus spp. strains. 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. γ-PGA, an anionic, water-soluble, biodegradable, non-toxic extra-cellular viscous material, is produced predominantly by Bacillus strains.
Ivanovics discovered γ-PGA as a Bacillus anthracis capsule, that was released into the medium while autoclaving the aged and autolyzed cells. 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 the food and pharmaceutical industries.
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. Cost-effective substrates and efficient strains are required for the production of the biopolymers commercially through microbial fermentation. Although α-PGA can be synthesized chemically and through biotransformation, these approaches are neither economic nor ecological.
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. The molecular weight of γ-PGA, which can range from 100 to 2500 kDa, significantly affect its chain length; as the molecular weight increases, so does the viscosity of the biopolymer, indicating a direct correlation between these two properties. Furthermore, the stability of the α-helical conformation of γ-PGA is maintained within a pH range 2.5 to 5.5, facilitating the formation of additional COO- groups 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.
Biopolymers have been 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 environmentally friendly methods over chemical-based polymers but are also easy to maintain and produce, and above all, they are cost-effective. The γ-PGA and its derivatives have become eminent biopolymers due to their widespread applications in the fields of food to drug discovery. The desirable physico-chemical properties like water solubility, water-holding capacity (WHC), and flowability make it a useful ingredient in food products over chemical-based ones. This review aims to focus on the current advances in the study of γ-PGA produced by various bacterial species, prominently by Bacillus sp. This study addresses γ-PGA biosynthesis, production, physico-chemical and functional characteristics, and multifaceted applications in the field of food, agriculture, healthcare, etc. This review emphasizes emerging opportunities for γ-PGA in industrial and therapeutic contexts.
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 B. subtilis and B. licheniformis) are generally employed. 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. α-PGA is chemically synthesized; however, γ-PGA is primarily produced by a wide variety of microbial species, particularly Bacillus sp., and unlike other proteins, it is not synthesized by ribosomes. Instead, it is generated as an extracellular polymer by Gram-positive bacteria and a few Gram-negative bacteria. B. subtilis and B. 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. 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.
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 homopolymers 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. The enzymes involved in the synthesis of γ-PGA have played a significant role in the development of production systems. 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. 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.
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 precursor for glutamic acid synthesis. The synthesis of γ-PGA occurs via four distinct stages such as racemization, polymerization, regulation, and degradation.
γ-PGA is typically composed of different variants: D-glutamate, L-glutamate, or a combination of both D- and L-enantiomers. 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. In fact, racemization reaction involves the conversion between the L- and D-forms. The racemization reaction in γ-PGA refers to the process by which the L-glutamic acid units convert to D-glutamic acid units, or vice versa, within the polymer chain. 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.
Research studies have reported the existence of four genes in Bacillus sp. (pgsB, C, A, and E) responsible for coding polyglutamate synthase (pgs), with ywsC, ywtAB, and capBCA identified as their Bacillus homologs in B. subtilis subspecies subtilis 168 and B. anthracis. 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 as the key components of the catalytic site. 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. Additionally, the presence of Zn2+ is essential for γ-PGA production in B. subtilis, requiring the involvement of pgsE. These studies demonstrated the intricate roles and interactions of multiple genes in the production of γ-PGA, highlighting the complex functions they perform collectively.
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. 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. 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. 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.The degradation of γ-PGA is facilitated by two enzymes: endo-γ-glutamyl peptidase and exo-γ-glutamyl peptidase. 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. 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. These enzymatic activities contribute to the degradation and metabolism of γ-PGA, influencing its molar mass and overall configuration.
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. 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. The fermentation process occurs in bioreactors under controlled conditions of temperature, pH, and oxygen supply. The microorganisms metabolize the nitrogen and carbon 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. 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 methods to enhance the expression of genes involved in γ-PGA synthesis, optimization of fermentation conditions, and the use of advanced bioprocess technologies.
Four different approaches are employed for the production of γ-PGA: microbial fermentation, peptide synthesis, chemical synthesis, and biotransformation. γ-PGA is a component commonly found in Japanese traditional food, specifically natto, which is prepared from fermented soybeans using Bacillus strains. Research has been done to study the nutritional requirements for γ-PGA production in order to improve its fermentation productivity. Bacillus strains are proficient in 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 g, is required by the bacteria for γ-PGA production. Different bacteria employ distinct γ-PGA production systems, indicating variations in the synthesis process. B. 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. 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. 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. amyloliquefaciens LL3 were cloned and co-expressed in E. coli JM 109 for the evaluation of γ-PGA productivity. The 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.
Studies report the use of different carbon sources for the efficient production of γ-PGA such as glucose, sucrose, xylose, starch, glycerol, and cane molasses. Glucose stood out as the most effective producer with 38.35 g/L yield. Parallelly, nitrogen sources like peptone, beef extract, yeast extract, soya peptone, soyabean meal, and corn steep liquor have also been used, where yeast extract was highlighted as the superior option, achieving a γ-PGA production 40.12 g/L. These studies underscore the significance of substrate selection, Hence, improving the production and its upscaling to industrial bioproduction.
γ-PGA from Bacillus spp.
Extensive research has been performed on the production and applications of γ-PGA (Table 1). PGA produced chemically results in the production of low molecular mass, i.e., 10 kDa, limiting its application. However, the γ-PGA (bacterial) varies from 10 to 100 kDa and may often reach as high as 10,000 kDa. 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) produces 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. There are two types of microorganisms involved in the production of γ-PGA, namely, L-glutamic acid-dependent and -independent bacteria. L-glutamic-acid-dependent bacteria include B. subtilis (chungkookjang), B. subtilis (natto) ATCC 15245, B. subtilis CGMCC 0833, B. licheniformis NK-03, and B. licheniformis 9945a. On the other hand, non-glutamic-acid-dependent bacteria include B. amyloliquefaciens LL3, B. subtilis C1, and B. subtilis C10.
| Name of Bacteria | Sources | Properties Studied |
| B. subtilis NRRL B-2612 | Devitalized wheat gluten | Solubility in water, molecular mass determination, viscosity |
| B. subtilis | Natto | Culture conditions, PGA analysis |
| B. subtilis ZJU-17 | Fermented bean curd | Effects of carbon sources and influence of nitrogen source on gamma polyglutamic acid production |
| B. subtilis | Natto | Application of γ-polyglutamic acid (Na+ form) in skincare products |
| B. subtilis DYU1 | Soil samples from a soy sauce manufacturing site | Flocculating activity and harmlessness to humans and environment |
| B. subtilis | Natto | Factors affecting production and agricultural applications |
| B. subtilis C10 | Sauce (from a local supermarket, China) | Isolation and characterisation of exogenous glutamic-acid-independent strain |
| B. subtilis | Natto | Rheology of biopolymers |
| B. subtilis | Nattokinase | High safety, simple production process, drug delivery system, excellent water solubility, biocompatibility, biodegradability |
| B. subtilis | Analysis of heavy metal distribution in soil | |
| B. subtilis ZC-5 | CICC, China | Solid-state fermentation, low cost substrates, environmental friendly process, reduced energy requirement and waste-water production |
| B. subtilis | Soil sample of the electroplating industry | Biodegradability, film-forming property, fibrogenicity, water-holding capacity |
| B. subtilis | Natto | Cryoprotective effects of γ-PGA, determination of dynamic rheological properties, Ca2+-ATPase activity, gel strength, salt-soluble protein content |
| B. subtilis (CGMCC17326) | Natto | Film forming property, reduced degree of browning in shiitake mushrooms |
| B. subtilis W-17 CICC 10260 | CICC | Use of γ-polyglutamic acid waste biomass |
| B. licheniformis A35 | Natto | Determination of amino acid |
| B. licheniformis | ATCC | Production optimization |
| B. licheniformis CCRC 12826 | CCRC, Taiwan | Production of biodegradable and harmless PGA |
| B. licheniformis WBL-3 (mutant of 9945) | ATCC | Effect of glycerol on cell growth and g-PGA production |
| B. licheniformis NCIM 2324 | NCIM | Molecular mass determination, amino acid analysis, total sugar content |
| B. licheniformis 9945 | ATCC | Production and purification and molecular size estimation |
| B. licheniformis A13 | Isolated from a tannery effluent | Optimization of PGA production |
| B. licheniformis NRC20 | Mine soil | Viscosity measurement, molecular mass determination, amino acid analysis |
| B. licheniformis ATCC 9945a | ATCC | Water absorption and solubility, graft content and efficiency, rheological behaviour |
| B. licheniformis | Applied Chemistry Research Center (Saltillo, Coahuila, Mexico) | Characterization of nanoparticles, encapsulation assays, bioactivity assays, in vitro release assays |
| B. licheniformis NBRC12107 | Fermented locust bean products | Characterization, tensile strength and porosity |
| B. licheniformis A14 | Marine sands | Microbially derived biopolymers are renewable in nature |
| B. subtilis and B. licheniformis | Reviewing different sources | Biopolymer rheology and viscosity–molecular mass correlation |
| B. subtilis and B. licheniformis | Chunkookjang | Chemical and microbial synthesis, application of PGA in medicine as a drug carrier and biological adhesives |
| B. subtilis and B. licheniformis | Natto | Biofilm formation, biosynthesis of PGA, genes involed, applications |
| B. subtilis ZJU-7 and B. licheniformis 9945a (NCIM 2324) | Reviewing many sources | Molecular mass determination, amino acid analysis, biodegradability, edibility and mmunogenicity |
| B. subtilis, B. licheniformis, and B. methylotrophicus | Natto and rhizosphere of pepper, cabbage, sweet corn, fenugreek leaves, barley, tomato, and sugarcane plants | Analysis to differentiate the monomeric and the polymeric forms of glutamic acid |
| Bacillus natto 20646 | Natto | PCR analysis |
| Bacillus sp. SJ-10 | Chungkookjang | Physicochemical properties and biofunctionality of PGA, molecular mass determination |
| Bacillus spp. FBL-2. | Chungkookjang | Optimization of medium components by central composite design (CCD) |
| Natrialba aegyptiaca and N. asiatica | Beach sand (Egypt) | Analysis of the extracellular polymer |
| B. amyloliquefaciens C06 | Mesophilic cheese starter | Molecular mass determination, UV scanning and amino acid analysis with paper chromatography |
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. Bajaj et al. (2009) 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), citric acid (15.2 g/L), ammonium sulfate (8.0 g/L), and L-glutamic acid (20 g/L), resulted in a yield of 26.12 g/L of γ-PGA. In comparison, the yield obtained with the basal medium was 5.27 g/L. The γ-PGA produced had a molecular mass of approximately 2.1 × 105 Da.
B. licheniformis Al3, a producer independent of exogenous glutamate, achieved a γ-PGA yield of 28.2 g/L in an optimized medium. The optimized medium consisted of glucose (50 g/L), NH4Cl (3 g/L), yeast extract (2 g/L), MgSO4.7H2O (0.8 g/L), NaCl (0.8 g/L), CaCl2.2H2O (0.00084 g/L), K2HPO4 (6.4 g/L), FeSO4.4H2O (0.006 g/L), 0.1 mL of trace element solution, and a culture volume of 23 mL. The Plackett-Burmann design was used up to 72 h after inoculation to assess the effects of different factors on γ-PGA production. 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. The same medium without agar was used for seed medium (50 mL) preparation and incubated at 37 ℃ for 24 h. The flasks were placed in a rotary shaker at 200 rpm. 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.
Bacillus subtilis
Bovarnick (1942) was the first to demonstrate that B. subtilis fermentation released the γ-PGA into the medium. 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) compared to the wild-type strain. Shih et al. 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 B. 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 min. 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.
Batch cultures of B. subtilis (natto) were conducted in a 5 L laboratory fermenter system, while a 600 L 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 ℃. The extracellular production of γ-PGA was observed with a molecular mass 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.
For B. 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 ℃ 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% K2HPO4, 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.
Bacillus anthracis and B. thuringiensis
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. 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. Hence, its cap gene responsible for the anchoring of γ-PGA onto its surface needs to be targeted to render B. anthracis immunogenic. 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.
While summarizing the production of γ-PGA from different Bacillus sp., it can be said that optimization strategies, such as nutrient supplementation (glutamate; glycerol; and ions like Na+, Ca2+, and Mn2+), fermentation conditions, and genetic modifications (targeting degradation enzymes), have significantly enhanced yields, reaching up to 101.1 g/L. Advanced purification methods, including ultrafiltration and ethanol precipitation, ensure high product purity (>95%) suitable for industrial applications. These advancements underscore γ-PGA’s growing potential in the biomedicine and food industries.
Structural and Physico-Chemical Properties of γ-PGA
γ-Polyglutamic acid (γ-PGA) exhibits diverse properties, including various conformational states, enantiomeric forms, and molecular mass. Its biodegradable, non-toxic, and non-immunogenic characteristics make it a valuable compound 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. 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. Physicochemical and functional characterization of γ-PGA molecules can be achieved using several modern techniques and instruments (Figure 2).
FTIR spectroscopy is the measurement of the procedure applied to record IR spectra. FTIR interferograms expose the functional groups in the purified γ-PGA, illustrating that it can be resolved by recognizing the specific peak values in the graphical values of the FTIR. FTIR spectroscopy was employed 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.
The γ-PGA synthesized by Bacillus spp. typically exhibits a high molecular mass ranging from 105 to 106 Da. Molecular mass estimation of γ-PGA has predominantly been conducted using gel permeation chromatography (GPC), employing various mobile phases and calibration against different standards. Bajestani et al. reported utilizing DEAE cellulose-52resin for running 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) with UV-detection at 216 nm depicting the chromatogram, followed by lyophilization. The molecular mass of γ-PGA as a heavy weight fraction was estimated to be 7.7 × 106 g/mol and 1.7 × 104 g/mol as the average molecular weight number. Birrer et al. followed another approach of chromatography, high-performance liquid chromatography (HPLC), as well as GPC to determine the number (Mn) and weight average molecular weights (Mw) along with polydispersity of γ-PGA. A calibration curve was constructed using narrow polydispersity pullulan standards, and the molecular weights M (Mw and Mn) of γ-PGA was calculated to be 22,000 g/mol and 266,000 g/mol, respectively.
HPLC is usually used in the analysis of amino acid composition. The γ-PGA hydrolysate chromatogram was detected at a position corresponding to D-glutamic acid having equal retention flow, and no peak corresponding to L-glutamic acid was detected. The result indicates that separated biocompatible γ-PGA contains D-glutamic acid residues the most.
Physico-Functional Properties
A substance capacity to retain moisture is water-holding capacity. γ-PGA is reported to have an excellent water-holding ability. 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. Additionally, the introduction of γ-PGA to sandy soils has been reported to have 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. The good water-binding capacity of γ-PGA results in an increase in moisture holding while reducing the oil uptake significantly. 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.
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 as well as foam stability of sponge cake paste, confirming the contribution of γ-PGA in delayed staling by. 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%).
The rheology studied by Zhang et al. focused on the rheological properties of γ-PGA produced by B. 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 to 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.
Conclusions
Dora PolyGlutamic Acid (γ-PGA) is produced through fermentation by Bacillus subtilis. γ-PGA is a homo-polymer of glutamic acid, it has excellent hydrophilic and high water absorption capacity. It can chelate trace elements in the soil, induce endogenous hormones and enhance plant resistance ability. Dora PolyGlutamic Acid also contains microbes, NPK, glucose, organic matter, etc. It is a great fertilizer synergist and a natural, organic, and environment-friendly plant nutrition enhancer.
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PolyGlutamic Acid (γ-PGA)
Dora PolyGlutamic Acid (γ-PGA) is a great fertilizer synergist and a natural, organic, and environment-friendly plant nutritions enhancer.