Optimization of fermentation conditions, purification and rheological properties of poly (γ-glutamic acid) produced by Bacillus subtilis 1006-3.

This study aimed to investigate the optimal fermentation condition, purification and rheological properties of extracellular polymers produced by Bacillus subtilis 1006-3. An optimum temperature of 30.2 °C, inoculation amount of 6.1%, and pH of 8.2 were determined via Response Surface Methodology. The result of amino acid analysis and gel electrophoresis indicated that the obtained polymer contained only glutamic acid, with a wide molecular weight range. This polymer was finally determined as γ-PGA by infrared spectroscopy. The γ-PGA solution displayed a behavior of pseudoplastic non-Newtonian fluid with shear thinning properties, which can be described by the Ostward-de Waele power law model. The apparent viscosity of γ-PGA solution increased with the increase in its concentration from 1% to 10%. The deviation in pH from neutral value, and the addition of NaCl or MgCl2 can reduce the apparent viscosity of γ-PGA solution, and it was more sensitive to Mg2+ than to Na+ addition. At the concentration of 4, 6, and 8%, γ-PGA solution showed predominantly viscous response in the range of 0.1-100 rad/s angular frequency (G″>G'). These results indicated the potential application of the γ-PGA as a thickening agent.

Efficient production of poly γ-d-glutamic acid from the bloom-forming green macroalgae, Ulva sp., by Bacillus sp. SJ-10.

Numerous studies on poly γ-d-glutamicacid (γ-PGA) production have investigated terrestrial renewable sources for reducing production costs, but there are no studies using waste marine resources so far. We aimed to develop a cost-effective production method of γ-d-PGA by Bacillus sp. SJ-10 using green macroalgae (Ulva sp.) as a major substrate without hydrolysis pretreatment. The SJ-10 was shown to not only cause immediate tissue degradation of the Ulva membrane but also grew well as a sole substrate. The γ-d-PGA yield was 6.29 ± 0.34 g/L under optimized conditions via the response surface method, and the produced γ-d-PGA had a thermal decomposition temperature of 310°C and molecular weight of 250-1780 kDa. The calculated cost efficiency for the final yield was 32% when compared with complex media. Therefore, the present study provided a strategy for promoting an ecofriendly and cost-effective means to produce γ-d-PGA via a marine renewable resource.

Purification of extra cellular poly-γ-glutamic acid as an antibacterial agent using anion exchange chromatography.

BLAST analysis of the 16S rRNA gene sequence for the newly isolated bacterium, revealed significant identity (99.5%) with Bacillus sonorensis [Ijadi Bajestani, M., et al., International Journal of Biological Macromolecules, 2017. 96: p. 100-110]. According to the literature review for closely related species of Bacillus sonorensis, the production of poly-γ-glutamic acid (γ-PGA) as an extra cellular biopolymer was investigated for the isolated bacteria which is deposited in IBRC (Iranian Biological Resource Center) as Bacillus sp. Strain M2 (IBRC-M11173). To determine if γ-PGA production by Bacillus sp. Strain M2 is glutamate dependent, it was grown on PGA medium, consisted of sodium glutamate. The results proved that γ-PGA production is highly dependent on glutamate component. In the following, the bioproduct has undergone different purification processes mainly consisting of dialysis, deproteinization and anion exchange chromatography. Based on the high-performance liquid chromatography (HPLC) results for ion chromatography effluents, 59% of the initial PGA in main solution was eluted via NaCl elution. Gel permeation chromatography (GPC) characterization analysis was accomplished to determine the polydispersity and γ-PGA molecular weight. Two major average molecular weights were distinguished; the heavy weight fraction of 7.7×106g/mol with polydispersity index of 1.73 and the other one with an average molecular weight number of 1.7×104g/mol and polydispersity index of 4.4. The antibacterial activity of the extracellular γ-PGA, as an anionic biopolymer, toward Staphylococcus aureus and E. coli, was assayed using the clinical and laboratory standards institute (CLSI) guidelines. For Staphylococcus aureus the minimum inhibitory concentration (MIC) value was about 34g/L while for E. coli this value reaches 53g/L.

Anti-herpes simplex virus 1 and immunomodulatory activities of a poly-γ- glutamic acid from Bacillus horneckiae strain APA of shallow vent origin.

Herpes simplex virus type 1 (HSV-1) is responsible of common and widespread viral infections in humans through the world, and of rare, but extremely severe, clinical syndromes in the central nervous system. The emergence of resistant strains to drugs actually in use encourages the searching for novel antiviral compounds, including those of natural origin. In this study, the recently described poly-γ-glutamic acid (γ-PGA-APA), produced by the marine thermotolerant Bacillus horneckiae strain APA, and previously shown to possess biological and antiviral activity, was evaluated for its anti-HSV-1 and immunomodulatory properties. The biopolymer hindered the HSV-1 infection in the very early phase of virus replication. In addition, the γ-PGA-APA was shown to exert low cytotoxicity and noticeable immunomodulatory activities towards TNF-α and IL-1ß gene expression. Moreover, the capacity to positively modulate the transcriptional activity of the cytokine genes was paired with increased level of activation of the transcription factor NF-kB by γ-PGA-APA. Overall, as non-cytotoxic biopolymer able to contribute in the antiviral defense against HSV-1, γ-PGA-APA could lead to the development of novel natural drugs for alternative therapies.

The poly-γ-d-glutamic acid capsule surrogate of the Bacillus anthracis capsule induces nitric oxide production via the platelet activating factor receptor signaling pathway.

The poly-γ-d-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, confers protection of the bacillus from phagocytosis and allows its unimpeded growth in the host. PGA capsules released from B. anthracis are associated with lethal toxin in the blood of experimentally infected animals and enhance the cytotoxic effect of lethal toxin on macrophages. In addition, PGA capsule itself activates macrophages and dendritic cells to produce proinflammatory cytokine such as IL-1ß, indicating multiple roles of PGA capsule in anthrax pathogenesis. Here we report that PGA capsule of Bacillus licheniformis, a surrogate of B. anthracis capsule, induces production of nitric oxide (NO) in RAW264.7 cells and bone marrow-derived macrophages. NO production was induced by PGA in a dose-dependent manner and was markedly reduced by inhibitors of inducible NO synthase (iNOS), suggesting iNOS-dependent production of NO. Induction of NO production by PGA was not observed in macrophages from TLR2-deficient mice and was also substantially inhibited in RAW264.7 cells by pretreatment of TLR2 blocking antibody. Subsequently, the downstream signaling events such as ERK, JNK and p38 of MAPK pathways as well as NF-κB activation were required for PGA-induced NO production. In addition, the induced NO production was significantly suppressed by treatment with antagonists of platelet activating factor receptor (PAFR) or PAFR siRNA, and mediated through PAFR/Jak2/STAT-1 signaling pathway. These findings suggest that PGA capsule induces NO production in macrophages by triggering both TLR2 and PAFR signaling pathways which lead to activation of NF-kB and STAT-1, respectively.

Construction of a Bacillus amyloliquefaciens strain for high purity levan production.

Bacillus amyloliquefaciens NK-1 has the potential to produce levan and poly-gamma-glutamic acid (γ-PGA) simultaneously. However, it is not possible to purify each single product from the same strain because the extraction process is identical. We deleted the pgs cluster (for γ-PGA synthesis) from the NK-1 strain and constructed a γ-PGA-deficient NK-ΔLP strain. Nuclear magnetic results showed that the NK-ΔLP strain could produce high purity levan product. However, its levan titer was only 1.96 g L(-1) in the basal medium. Single-factor experimental and response surface methodology was used to optimize the culture condition, leading to levan titer of 13.9 and 22.6 g L(-1) in flask culture and in a 5-L bioreactor, respectively. The levan purity can reach to 92.7% after 48 h cultivation. Furthermore, the relationship between levanase (LevB) and levan molecular weight was studied. The results showed that LevB resulted in the production of low molecular weight levan and its expression level determined the ratio of high and low molecular weight levan. We also deleted the sac cluster (for levan synthesis) from the NK-1 strain and constructed a levan-deficient NK-L strain. The NK-L strain exhibited increased purity of γ-PGA product from 79.5 to 91.2%.

Purification and characterization of gamma poly glutamic acid from newly Bacillus licheniformis NRC20.

γ-poly glutamic acid (γ-PGA) has received considerable attention for pharmaceutical and biomedical applications. γ-PGA from the newly isolate Bacillus licheniformis NRC20 was purified and characterized using diffusion distance agar plate, mass spectrometry and thin layer chromatography. All analysis indicated that γ-PGA is a homopolymer composed of glutamic acid. Its molecular weight was determined to be 1266 kDa. It was composed of L- and D-glutamic acid residues. An amplicon of 3050 represents the γ-PGA-coding genes was obtained, sequenced and submitted in genbank database. Its amino acid sequence showed high similarity with that obtained from B. licheniformis strains. The bacterium NRC 20 was independent of L-glutamic acid but the polymer production enhanced when cultivated in medium containing L-glutamic acid as the sole nitrogen source. Finally we can conclude that γ-PGA production from B. licheniformis NRC20 has many promised applications in medicine, industry and nanotechnology.

Immunomodulatory effect of poly-γ-glutamic acid derived from Bacillus subtilis on natural killer dendritic cells.

Bacillus subtilis-derived poly-γ-glutamic acid (γPGA) stimulates dendritic cells (DCs) to produce IL12, leading to CD4(+) T cell differentiation toward the Th1 phenotype, but DCs consist of heterogeneous subpopulations with a variety of immune functions. Among these, natural killer dendritic cells (NKDCs) play an important role in anti-tumor immune responses. Herein, we demonstrate the role of NKDCs in γPGA-meditated anti-tumor immune responses. NK1.1(+) CD11c(+) NKDCs were stimulated upon γPGA stimulation in vitro and in vivo to up-regulate lymphocyte activation markers, MHC class I and II, and co-stimulatory molecules. In particular, NKDCs were activated by γPGA to produce IFNγ and TNFα, like NK cells, as well as IL12, like DCs, implying that NKDCs have unique and multifunctional roles. Importantly, NKDCs stimulated by γPGA conferred stronger anti-tumor effects in mice and showed increased cytotoxicity against various tumor cell lines in vitro. In conclusion, NKDCs are one of the key players in anti-tumor immunity induced by γPGA.

Poly-γ-glutamate from Bacillus subtilis inhibits tyrosinase activity and melanogenesis.

Poly-γ-glutamate (γ-PGA) has been considered as one of the most promising biomaterials with a wide range of applications, but there has been no report that directly shows the anti-tyrosinase and anti-melanogenesis properties of γ-PGA. In the present study, we investigated the inhibitory effects of γ- PGA with low molecular weight (Mw; lγ-PGA) and high Mw (hγ-PGA) on mushroom tyrosinase and murine tyrosinase activities and on melanogenesis in B16 melanoma cells. First, we showed that both lγ-PGA and hγ-PGA could effectively inhibit mushroom tyrosinase activities including monophenolase and diphenolase activities in a dose-dependent manner. Second, both lγ-PGA and hγ-PGA showed strong anti-tyrosinase activity and anti-melanogenesis in B16 melanoma cells. Third, both lγ-PGA and hγ-PGA inhibited forskolin-induced tyrosinase activity and melanogenesis by decreasing the levels of intracellular reactive oxygen species and nitric oxide while increasing the catalase activity in B16 cells. This is the first report on the anti-melanogenesis effect of γ-PGA, which suggests that γ-PGA could have a potential in the cosmetic skin whitening business, therapeutic applications and the food industry.

Fabrication of poly(γ-glutamic acid) monolith by thermally induced phase separation and its application.

Monoliths are functional porous materials with a three-dimensional continuous interconnected pore structure in a single piece. A monolith with uniform shape based on poly(γ-glutamic acid) (PGA) has been prepared via a thermally induced phase separation technique using a mixture of dimethyl sulfoxide, water, and ethanol as solvent. The morphology of the obtained monolith was observed by scanning electron microscopy and the surface area of the monolith was evaluated by the Brunauer Emmett Teller method. The effects of fabrication parameters such as the concentration and molecular mass of PGA and the solvent composition have been systematically investigated. The PGA monolith was cross-linked with hexamethylene diisocyanate to produce the water-insoluble monolith. The addition of sodium chloride to the phase separation solvent affected the properties of the cross-linked monolith. The swelling ratio of the cross-linked monolith toward aqueous solutions depended on the buffer pH as well as the monolith fabrication condition. Copper(II) ion was efficiently adsorbed on the cross-linked PGA monolith, and the obtained copper-immobilized monolith showed strong antibacterial activity for Escherichia coli. By combination of the characteristic properties of PGA (e.g., high biocompatibility and biodegradability) and the unique features of monoliths (e.g., through-pore structure, large surface area, and high porosity with small pore size), the PGA monolith possesses large potentials for various industrial applications in the biomedical, environmental, analytical, and separation fields.

High-molecular-weight poly-gamma-glutamate protects against hypertriglyceridemic effects of a high-fructose diet in rat.

We studied the effects of 2 different dosages of highmolecular- weight poly-γ-glutamic acid (hm γ-PGA) derived from Bacillus subtilis chungkookjang on lipid metabolism in a high-fructose diet-induced hypertriglyceridemic animal model. For 4 weeks, rats were fed either AIN-93 diet (normal control, NC; n = 10) or modified AIN-93 diet in which cornstarch was substituted with 63% fructose (n = 30) to induce hypertriglyceridemia. After 4 weeks, the hypertriglyceridemic rats were treated with daily oral doses of 0 mg (hypertriglyceridemic control, HC), 2.5 mg (hypertriglyceridemic, low hm γ-PGA, HL), or 5 mg·kg·bw(-1)·d(-1) (hypertriglyceridemic, high hm γ-PGA, HH) hm γ-PGA for 4 weeks. The HL and HH groups exhibited significantly lower levels of serum triglyceride, total cholesterol, LDL cholesterol, and free fatty acids than the HC group. The administration of hm γ-PGA reduced serum ALT and AST levels. The activities of lipogenic enzymes such as hepatic malic enzyme and glucose-6-phosphate dehydrogenase as well as glucose-6-phosphate dehydrogenase mRNA expression were significantly decreased by hm γ-PGA administration (p < 0.05). These results indicate that hm γ- PGA has an anti-hypertriglyceridemic effect in highfructose diet-induced hypertriglyceridemic rats.

Heterogenous expression of poly-gamma-glutamic acid synthetase complex gene of Bacillus licheniformis WBL-3.

Bacillus licheniformis WBL-3, one of poly-gamma-glutamic acid (gamma-PGA) producers, depends on the existence of glutamate in the medium. In this paper, gamma-PGA synthetase complex gene (pgsBCA) was cloned from Bacillus licheniformis WBL-3. pgsBCA gene of B. licheniformis WBL-3 was highly homologous with pgsBCA gene of B. licheniformis 14580. The similarity was 97%, but the similarity of pgsBCA gene between B. licheniformis WBL-3 and Bacillus subtilis IF03336 was only 74%. However, when pgsBCA was expressed in Escherichia coli, the E. coli clone produced gamma-PGA extracellularly. The yield of gamma-PGA was 8.624 g/l. This result infers that B. licheniformis and B. subtilis has the similar gamma-PGA biosynthesis mechanism, namely, glutamic acid is catalyzed by an ATP-dependent amide ligase to synthesize gamma-PGA.

Poly (glutamic acid)--an emerging biopolymer of commercial interest.

Poly (γ-glutamic acid) (PGA) is water-soluble, anionic, biodegradable, and edible biopolymer produced by Bacillus subtilis. It has multifarious potential applications in foods, pharmaceuticals, healthcare, water treatment and other fields. The production of PGA has already been established on the industrial scale. Various studies regarding the fermentative production, downstream processing and characterization of PGA have been reported in the literature. This review provides updated information on fermentative production of PGA by various bacterial strains and effect of fermentation conditions and media component on production of PGA in submerged as well as solid state fermentation. Information on the application of genetic engineering for enhancement of yield of PGA, kinetic studies for production of PGA in submerged fermentation and recovery and purification of PGA is included. An attempt has also been made to review the current and potential applications of PGA. This review may contribute to further development of this commercially and academically interesting biopolymer.

A novel Method for the selective recovery and purification of gamma-polyglutamic acid from Bacillus licheniformis fermentation broth.

Microbially produced gamma-polyglutamic acid (gamma-PGA) is a commercially important biopolymer with many applications in biopharmaceutical, food, cosmetic and waste-water treatment industries. Owing to its increasing demand in various industries, production of gamma-PGA is well documented in the literature, however very few methods have been reported for its recovery. In this paper, we report a novel method for the selective recovery and purification of gamma-PGA from cell-free fermentation broth of Bacillus licheniformis. The cell-free fermentation broth was treated with divalent copper ions, resulting in the precipitation of gamma-PGA, which was collected as a pellet by centrifugation. The pellet was resolubilized and dialyzed against de-ionized water to obtain the purified gamma-PGA biopolymer. The efficiency and selectivity of gamma-PGA recovery was compared with ethanol precipitation method. We found that 85% of the original gamma-PGA content in the broth was recovered by copper sulfate-induced precipitation, compared to 82% recovery by ethanol precipitation method. Since ethanol is a commonly used solvent for protein precipitation, the purity of gamma-PGA precipitate was analyzed by measuring proteins that co-precipitated with gamma-PGA. Of the total proteins present in the broth, 48% proteins were found to be co-precipitated with gamma-PGA by ethanol precipitation, whereas in copper sulfate-induced precipitation, only 3% of proteins were detected in the final purified gamma-PGA, suggesting that copper sulfate-induced precipitation offers better selectivity than ethanol precipitation method. Total metal content analysis of the purified gamma-PGA revealed the undetectable amount of copper ions, whereas other metal ions detected were in low concentration range. The purified gamma-PGA was characterized using infrared spectroscopy.

Phylogeny of gamma-polyglutamic acid-producing Bacillus strains isolated from a fermented locust bean product manufactured in West Africa.

Twenty-five Bacillus strains capable of producing gamma-polyglutamic acid (PGA) were isolated from fermented locust bean products manufactured in the savanna area of Ghana. To clarify the phylogeny of these PGA-producing strains, phylogenetic analyses based on sequences of 16S rDNA, rpoB (RNA polymerase beta-subunit) and fus (elongation factor G) genes were performed. A phylogenetic tree based on 16S rDNA indicated that ten isolates were clustered in the same group of Bacillus subtilis. Another ten isolates were located in the cluster of B. amyloliquefaciens, and the remaining isolates were identified as B. pumilus (three isolates) and B. licheniformis (two isolates), respectively. Phylogenetic trees based on the partial sequences of rpoB and fus genes were similar to the phylogeny based on 16S rDNA sequences. Thirty-four strains in 27 species belonging to the genus Bacillus and its neighbors were also investigated for PGA production. It was found that PGA was produced by B. amyloliquefaciens NBRC 14141 and NBRC 15535(T), B. atrophaeus NBRC 15539(T), B. licheniformis NBRC 12107, B. mojavensis NBRC 15718(T), B. pumilus NBRC 12094, B. subtilis NBRC 16449, and Lysinibacillus sphaericus NBRC 3525. Except for L. sphaericus, the above Bacillus species are very closely related in phylogeny, indicating that PGA-producing Bacillus strains constitute a cluster.

Enhanced production of poly (gamma-glutamic acid) from Bacillus licheniformis NCIM 2324 in solid state fermentation.

This work reports on the optimization of PGA production by Bacillus licheniformis NCIM 2324 in solid state fermentation (SSF). In the first step, the one factor-at-a-time method was used to investigate the effect of solid substrates, initial moisture content, pH, and additional carbon and nitrogen source on PGA production; subsequently, response surface methodology (RSM) was used to establish the optimum concentrations of the key nutrients for higher PGA production. In the second step, the effects of amino acids and TCA cycle intermediates on the production of PGA were studied. The final optimized medium gave a maximum yield of 98.64 +/- 1.61 mg gds(-1) of PGA, which is significantly higher than that reported in the literature.

Tubulin polyglutamylase enzymes are members of the TTL domain protein family.

Polyglutamylation of tubulin has been implicated in several functions of microtubules, but the identification of the responsible enzyme(s) has been challenging. We found that the neuronal tubulin polyglutamylase is a protein complex containing a tubulin tyrosine ligase-like (TTLL) protein, TTLL1. TTLL1 is a member of a large family of proteins with a TTL homology domain, whose members could catalyze ligations of diverse amino acids to tubulins or other substrates. In the model protist Tetrahymena thermophila, two conserved types of polyglutamylases were characterized that differ in substrate preference and subcellular localization.

Efficient recovery of gamma-poly (glutamic acid) from highly viscous culture broth.

An efficient strategy for the separation and recovery of gamma-polyglutamic acid (gamma-PGA) from highly viscous broth was developed. This strategy was divided into two processes: The first was to separate gamma-PGA from highly viscous culture broth; the second was to concentrate gamma-PGA solution by ultrafiltration for the reduction of the amount of alcohol required during recovery process with precipitation. By lowering the pH value of culture broth to 3, the viscosity of culture broth and the zeta potential of cell could be reduced to a sixth of the original value at 35 degrees C and a third, respectively. After the acidification of culture broth the energy demand for the separation of gamma-PGA from culture broth by centrifugation could be reduced to 17% of that without it when the centrifugal force was 22,000g. The amount of alcohol required for precipitation could be reduced to a fourth of that generally used without concentration by concentrating 20 g gamma-PGA/L solution to 60 g gamma-PGA/L at pH 5 by ultrafiltration with hollow-fiber membrane cartridge (MWCO 500,000).

Synthesis of paucidisperse poly(gamma-benzyl-alpha,L-glutamate) oligomers and star polymers with rigid arms.

The synthesis of highly uniform gamma-benzyl-alpha,L-glutamate (BLG) oligomers via a convergent solution phase approach is reported. BLG oligomers were produced with designed lengths of 4, 8, 12, and 16 as a first step to production of BLG-4-mer and BLG-8-mer rod stars. The star oligomers were purified by size-exclusion chromatography and reversed phase HPLC, and characterized by MALDI-TOF mass spectrometry and reversed phase HPLC. These star-shaped BLG oligomers could be used as initiators for growing larger stars.