Enhanced poly-γ-glutamic acid synthesis in Corynebacterium glutamicum by reconstituting PgsBCA complex and fermentation optimization.

Previously, a novel Corynebacterium glutamicum strain for the de novo biosynthesis of tailored poly-γ-glutamic acid (γ-PGA) has been constructed by our group. The strain was based on the γ-PGA synthetase complex, PgsBCA, which is the only polyprotein complex responsible for γ-PGA synthesis in Bacillus spp. In the present study, PgsBCA was reconstituted and overexpressed in C. glutamicum to further enhance γ-PGA synthesis. First, we confirmed that all the components (PgsB, PgsC, and PgsA) of γ-PGA synthetase derived from B. licheniformis are necessary for γ-PGA synthesis, and γ-PGA was detected only when PgsB, PgsC, and PgsA were expressed in combination in C. glutamicum. Next, the expression level of each pgsB, pgsC, and pgsA was tuned in order to explore the effect of expression of each of the γ-PGA synthetase subunits on γ-PGA production. Results showed that increasing the transcription levels of pgsB or pgsC and maintaining a medium-level transcription level of pgsA led to 35.44% and 76.53% increase in γ-PGA yield (γ-PGA yield-to-biomass), respectively. Notably, the expression level of pgsC had the greatest influence (accounting for 68.24%) on γ-PGA synthesis, followed by pgsB. Next, genes encoding for PgsC from four different sources (Bacillus subtilis, Bacillus anthracis, Bacillus methylotrophicus, and Bacillus amyloliquefaciens) were tested in order to identify the influence of PgsC-encoding orthologues on γ-PGA production, but results showed that in all cases the synthesis of γ-PGA was significantly inhibited. Similarly, we also explored the influence of gene orthologues encoding for PgsB on γ-PGA production, and found that the titer increased to 17.14 ± 0.62 g/L from 8.24 ± 0.10 g/L when PgsB derived from B. methylotrophicus replaced PgsB alone in PgsBCA from B. licheniformis. The resulting strain was chosen for further optimization, and we achieved a γ-PGA titer of 38.26 g/L in a 5 L fermentor by optimizing dissolved oxygen level. Subsequently, by supplementing glucose, γ-PGA titer increased to 50.2 g/L at 48 h. To the best of our knowledge, this study achieved the highest titer for de novo production of γ-PGA from glucose, without addition of L-glutamic acid, resulting in a novel strategy for enhancing γ-PGA production.

Genomic characterization and related functional genes of γ- poly glutamic acid producing Bacillus subtilis.

γ- poly glutamic acid (γ-PGA), a high molecular weight polymer, is synthesized by microorganisms and secreted into the extracellular space. Due to its excellent performance, γ-PGA has been widely used in various fields, including food, biomedical and environmental fields. In this study, we screened natto samples for two strains of Bacillus subtilis N3378-2at and N3378-3At that produce γ-PGA. We then identified the γ-PGA synthetase gene cluster (PgsB, PgsC, PgsA, YwtC and PgdS), glutamate racemase RacE, phage-derived γ-PGA hydrolase (PghB and PghC) and exo-γ-glutamyl peptidase (GGT) from the genome of these strains. Based on these γ-PGA-related protein sequences from isolated Bacillus subtilis and 181 B. subtilis obtained from GenBank, we carried out genotyping analysis and classified them into types 1-5. Since we found B. amyloliquefaciens LL3 can produce γ-PGA, we obtained the B. velezensis and B. amyloliquefaciens strains from GenBank and classified them into types 6 and 7 based on LL3. Finally, we constructed evolutionary trees for these protein sequences. This study analyzed the distribution of γ-PGA-related protein sequences in the genomes of B. subtilis, B. velezensis and B. amyloliquefaciens strains, then the evolutionary diversity of these protein sequences was analyzed, which provided novel information for the development and utilization of γ-PGA-producing strains.

Untargeted metabolomics revealed the effect of soybean metabolites on poly(γ-glutamic acid) production in fermented natto and its metabolic pathway.

BACKGROUND: Natto mucus is mainly composed of poly(γ-glutamic acid) (γ-PGA), which affects the sensory quality of natto and has some effective functional activities. The soybean metabolites that cause different γ-PGA contents in different fermented natto are unclear. RESULTS: In this study, we use untargeted metabolomics to analyze the metabolites of high-production γ-PGA natto and low-production γ-PGA natto and their fermented substrate soybean. A total of 257 main significantly different metabolites with the same trend among the three comparison groups were screened, of which 114 were downregulated and 143 were upregulated. Through the enrichment of metabolic pathways, the metabolic pathways with significant differences were purine metabolism, nucleotide metabolism, fructose and mannose metabolism, anthocyanin biosynthesis, isoflavonoid biosynthesis and the pentose phosphate pathway. CONCLUSION: For 114 downregulated main significantly different metabolites with the same trend among the three comparison groups, Bacillus subtilis (natto) may directly decompose them to synthesize γ-PGA. Adding downregulated substances before fermentation or cultivating soybean varieties with the goal of high production of such substances has a great effect on the production of γ-PGA by natto fermentation. The enrichment analysis results showed the main pathways affecting the production of γ-PGA by Bacillus subtilis (natto) using soybean metabolites, which provides a theoretical basis for the production of γ-PGA by soybean and promotes the diversification of natto products. © 2023 Society of Chemical Industry.

Effects of Fe2+ addition to sugarcane molasses on poly-γ-glutamic acid production in Bacillus licheniformis CGMCC NO. 23967.

BACKGROUND: Poly-γ-glutamic acid (γ-PGA) is biodegradable, water-soluble, environment-friendly, and edible. Consequently, it has a variety of industrial applications. It is crucial to control production cost and increase output for industrial production γ-PGA. RESULTS: Here γ-PGA production from sugarcane molasses by Bacillus licheniformis CGMCC NO. 23967 was studied in shake-flasks and bioreactors, the results indicate that the yield of γ-PGA could reach 40.668 g/L in a 5L stirred tank fermenter. Further study found that γ-PGA production reached 70.436 g/L, γ-PGA production and cell growth increased by 73.20% and 55.44%, respectively, after FeSO4·7H2O was added. Therefore, we investigated the metabolomic and transcriptomic changes following FeSO4·7H2O addition. This addition resulted in increased abundance of intracellular metabolites, including amino acids, organic acids, and key TCA cycle intermediates, as well as upregulation of the glycolysis pathway and TCA cycle. CONCLUSIONS: These results compare favorably with those obtained from glucose and other forms of biomass feedstock, confirming that sugarcane molasses can be used as an economical substrate without any pretreatment. The addition of FeSO4·7H2O to sugarcane molasses may increase the efficiency of γ-PGA production in intracellular.

Oil-Contaminated Soil Remediation with Biodegradation by Autochthonous Microorganisms and Phytoremediation by Maize (Zea mays).

Biological methods are currently the most commonly used methods for removing hazardous substances from land. This research work focuses on the remediation of oil-contaminated land. The biodegradation of aliphatic hydrocarbons and PAHs as a result of inoculation with biopreparations B1 and B2 was investigated. Biopreparation B1 was developed on the basis of autochthonous bacteria, consisting of strains Dietzia sp. IN118, Gordonia sp. IN101, Mycolicibacterium frederiksbergense IN53, Rhodococcus erythropolis IN119, Rhodococcus globerulus IN113 and Raoultella sp. IN109, whereas biopreparation B2 was enriched with fungi, such as Aspergillus sydowii, Aspergillus versicolor, Candida sp., Cladosporium halotolerans, Penicillium chrysogenum. As a result of biodegradation tests conducted under ex situ conditions for soil inoculated with biopreparation B1, the concentrations of TPH and PAH were reduced by 31.85% and 27.41%, respectively. Soil inoculation with biopreparation B2 turned out to be more effective, as a result of which the concentration of TPH was reduced by 41.67% and PAH by 34.73%. Another issue was the phytoremediation of the pre-treated G6-3B2 soil with the use of Zea mays. The tests were carried out in three systems (system 1-soil G6-3B2 + Zea mays; system 2-soil G6-3B2 + biopreparation B2 + Zea mays; system 3-soil G6-3B2 + biopreparation B2 with γ-PGA + Zea mays) for 6 months. The highest degree of TPH and PAH reduction was obtained in system 3, amounting to 65.35% and 60.80%, respectively. The lowest phytoremediation efficiency was recorded in the non-inoculated system 1, where the concentration of TPH was reduced by 22.80% and PAH by 18.48%. Toxicological tests carried out using PhytotoxkitTM, OstracodtoxkitTM and Microtox® Solid Phase tests confirmed the effectiveness of remediation procedures and showed a correlation between the concentration of petroleum hydrocarbons in the soil and its toxicity. The results obtained during the research indicate the great potential of bioremediation practices with the use of microbial biopreparations and Zea mays in the treatment of soils contaminated with petroleum hydrocarbons.

Poly-γ-glutamic acid promoted maize root development by affecting auxin signaling pathway and the abundance and diversity of rhizosphere microbial community.

BACKGROUND: The root systems of higher plants play an important role in plant growth and development. In our present study, it was found that poly-γ-glutamic acid (γ-PGA), an environmentally friendly biomacromolecule, significantly improved root development in maize. RESULTS: After treatment with γ-PGA for 7 days, the fresh weight of maize roots was significantly increased and the differences between γ-PGA treated group and control group were mainly caused by the number (higher by 71.87% compared to the control) and length of lateral roots. RNAseq and RT-PCR analyses showed that γ-PGA treatment upregulated the expression of genes related to the synthesis of auxins and auxin signal in maize roots. In addition, γ-PGA promoted the accumulation of plant growth-promoting bacteria, such as Azospirillum, Azohydromonas, Ramlibacter, and Sphingobium (Proteobacteria), Streptomyces (Actinobacteria), Parasegetibacter (Bacteroidetes), and Gemmatimonas (Gemmatimonadetes) in rhizosphere soil and the secretion of auxins. The results of this study deepened our understanding of the effects and mechanism of γ-PGA on maize root development, and as well as highlighted the possibility of using γ-PGA to improve crop growth and soil environment. CONCLUSIONS: γ-PGA promotes early growth and development of maize roots by inducing the secretion and accumulation of auxin in roots and in rhizosphere soil, and increasing the abundance of plant growth promoting bacteria.

Poly-γ-glutamic acid enhanced the drought resistance of maize by improving photosynthesis and affecting the rhizosphere microbial community.

BACKGROUND: Compared with other abiotic stresses, drought stress causes serious crop yield reductions. Poly-γ-glutamic acid (γ-PGA), as an environmentally friendly biomacromolecule, plays an important role in plant growth and regulation. RESULTS: In this project, the effect of exogenous application of γ-PGA on drought tolerance of maize (Zea mays. L) and its mechanism were studied. Drought dramatically inhibited the growth and development of maize, but the exogenous application of γ-PGA significantly increased the dry weight of maize, the contents of ABA, soluble sugar, proline, and chlorophyll, and the photosynthetic rate under severe drought stress. RNA-seq data showed that γ-PGA may enhance drought resistance in maize by affecting the expression of ABA biosynthesis, signal transduction, and photosynthesis-related genes and other stress-responsive genes, which was also confirmed by RT-PCR and promoter motif analysis. In addition, diversity and structure analysis of the rhizosphere soil bacterial community demonstrated that γ-PGA enriched plant growth promoting bacteria such as Actinobacteria, Chloroflexi, Firmicutes, Alphaproteobacteria and Deltaproteobacteria. Moreover, γ-PGA significantly improved root development, urease activity and the ABA contents of maize rhizospheric soil under drought stress. This study emphasized the possibility of using γ-PGA to improve crop drought resistance and the soil environment under drought conditions and revealed its preliminary mechanism. CONCLUSIONS: Exogenous application of poly-γ-glutamic acid could significantly enhance the drought resistance of maize by improving photosynthesis, and root development and affecting the rhizosphere microbial community.

Poly-γ-glutamic acid/Alum adjuvanted pH1N1 vaccine-immunized aged mice exhibit a significant increase in vaccine efficacy with a decrease in age-associated CD8+ T cell proportion in splenocytes.

BACKGROUND: Highly contagious respiratory diseases caused by viral infections are a constantly emerging threat, particularly the elderly with the higher risk of developing serious complications. Vaccines are the best strategy for protection against influenza-related diseases. However, the elderly has lower vaccine efficacy than young population and the age-driven decline of the influenza vaccine efficacy remains unresolved. OBJECTIVES: This study investigates the effect of an adjuvant, poly-γ-glutamic acid and alum (PGA/Alum) on vaccine efficacy in aged mice (18-months) and its mechanism is investigated using ovalbumin as a model antigen and a commercial pandemic H1N1 (pH1N1) flu vaccine. Antigen trafficking, dendritic cell (DC) activation, and the DC-mediated T cell activation were analyzed via in vivo imaging and flow cytometry. Antigen-specific humoral and cellular immune responses were evaluated in sera and splenocytes from the vaccinated mice. Also, we analyzed gene expression profiles of splenocytes from the vaccinated mice via single-cell transcriptome sequencing and evaluated the protective efficacy against pH1N1 virus challenge. RESULTS: Aged mice had lower antigen trafficking and DC activation than younger mice (6-weeks), which was ameliorated by PGA/Alum with increased antigen uptake and DC activation leading to improved antigen-specific IFN-γ+CD8+ T lymphocyte frequencies higher in the vaccinated aged mice, to a similar extent as PGA/Alum adjuvanted vaccine-immunized young mice. The results of single-cell transcriptome sequencing display that PGA/Alum also reduced the proportion of age-associated CD8+ T cell subsets and gene levels of inhibitory regulators in CD8+ T cells, which may play a role in the recovery of CD8+ T cell activation. Finally, PGA/Alum adjuvanted pH1N1 vaccine-immunized aged mice were completely protected (100% survival) compared to aged mice immunized with vaccine only (0% survival) after pH1N1 virus challenge, akin to the efficacy of the vaccinated young mice (100% survival). CONCLUSIONS: PGA/Alum adjuvanted pH1N1 vaccine-immunized aged mice showed a significant increase in vaccine efficacy compared to aged mice administered with vaccine only. The enhanced vaccine efficacy by PGA/Alum is associated with significant increases of activation of DCs and effector CD8+ T cells and a decrease in age-associated CD8+ T cell proportion of splenocytes. Collectively, PGA/Alum adjuvanted flu vaccine may be a promising vaccine candidate for the elderly.

Enhanced production of poly-γ-glutamic acid by Bacillus licheniformis ATCC 9945a using simultaneous pulse-feedings of citrate and glutamate.

Poly-γ-glutamic acid (γ-PGA) is a versatile biopolymer with widespread applications in the food, pharmaceutical, and medical industries. One of the main challenges in expanding γ-PGA industrial applications is the high cost of production. Developing an efficient and low-cost fermentation process such as bacterial cultivation with pulsed feeding can significantly reduce production costs. Thus, initially, a new pulsed-feeding strategy of citrate and glutamate was developed for γ-PGA production enhancement in the fed-batch culture of Bacillus licheniformis ATCC 9945a. Then, the effects of pulse number, feeding amount, feeding times, the addition time of calcium and manganese solutions, the pH of the added citrate solution, and the concentration of feed stock solutions of pulse-feeds on γ-PGA production were investigated. Under optimal conditions: feeding two pulses at 8 and 24 hours of culture, 20 g citrate and glutamate per liter of culture medium per pulse (about 52 mL of each of citrate and glutamate feeding solutions prepared with a concentration of 384 g/L by adding distilled water) about 88 ± 4 g/L of γ-PGA was obtained. It is one of the highest values ever reported for γ-PGA production with Bacillus licheniformis ATCC 9945a, of course with a much simpler process than the other fed-batch processes.

Study on the mechanism of production of γ-PGA and nattokinase in Bacillus subtilis natto based on RNA-seq analysis.

Poly-γ-glutamic acid (γ-PGA) and nattokinase (NK) are the main substances produced by Bacillus subtilis natto in solid-state fermentation and have wide application prospects. We found that our strains had higher activity of nattokinase when soybeans were used as substrate to increase the yield of γ-PGA. Commercial production of γ-PGA and nattokinase requires an understanding of the mechanism of co-production. Here, we obtained the maximum γ-PGA yield (358.5 g/kg, w/w) and highest activity of NK during fermentation and analyzed the transcriptome of Bacillus subtilis natto during co-production of γ-PGA and NK. By comparing changes in expression of genes encoding key enzymes and the metabolic pathways associated with the products in genetic engineering, the mechanism of co-production of γ-PGA and nattokinase can be summarized based on RNA-seq analysis. This study firstly provides new insights into the mechanism of co-production of γ-PGA and nattokinase by Bacillus subtilis natto and reveals potential molecular targets to promote the co-production of γ-PGA and nattokinase.

Poly-γ-glutamic acid production by simultaneous saccharification and fermentation using corn straw and its fertilizer synergistic effect evaluation.

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

Biosynthesis of poly-γ-glutamic acid in Escherichia coli by heterologous expression of pgsBCAE operon from Bacillus.

AIMS: Poly-γ-glutamic acid (γ-PGA) is an excellent water-soluble biosynthesis material. To confirm the rate-limiting steps of γ-PGA biosynthesis pathway, we introduced a heterologous Bacillus strain pathway and employed an enzyme-modulated dismemberment strategy in Escherichia coli. METHODS AND RESULTS: In this study, we heterologously introduced the γ-PGA biosynthesis pathway of two laboratory-preserved strains-Bacillus amyloliquefaciens FZB42 and Bacillus subtilis 168 into E. coli, and compared their γ-PGA production levels. Next, by changing the plasmid copy numbers and supplying sodium glutamate, we explored the effects of gene expression levels and concentrations of the substrate l-glutamic acid on γ-PGA production. We finally employed a two-plasmid induction system using an enzyme-modulated dismemberment of pgsBCAE operon to confirm the rate-limiting genes of the γ-PGA biosynthesis pathway. CONCLUSION: Through heterologously over-expressing the genes of the γ-PGA biosynthesis pathway and exploring gene expression levels, we produced 0·77 g l-1 γ-PGA in strain RSF-EBCAE(BS). We also confirmed that the rate-limiting genes of the γ-PGA biosynthesis pathway were pgsB and pgsC. SIGNIFICANCE AND IMPACT OF THE STUDY: This work is beneficial to increase γ-PGA production and study the mechanism of γ-PGA biosynthesis enzymes.

Enhancing the production of poly-γ-glutamate in Bacillus subtilis ZJS18 by the heat- and osmotic shock and its mechanism.

Poly-γ-glutamate (γ-PGA) is a natural macromolecule peptide, and is widely used in the food, medicine, and pharmaceutical industries. In this study, heat- and osmotic shock were used to improve the production of γ-PGA in Bacillus subtilis ZJS18, and its molecular mechanism was explored. The results indicated that the heat- and osmotic shock significantly promoted the production of γ-PGA owing to the stress response of B. subtilis cells to adverse environment. The highest concentrations of γ-PGA reached 14.53 and 15.98 g/l under heat- and osmotic shock, respectively. The activities of five enzymes related to the metabolism of the endogenous glutamate were determined and analyzed. It was found that the activities of glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, glutamate dehydrogenase and glutamate synthase were significantly altered during heat- and osmotic shock, while the activity of α-ketoglutarate dehydrogenase only showed a little alteration. This study provides a basis for the industrial production and use of γ-PGA, and for understanding its biosynthetic mechanism in B. subtilis ZJS18.

Tailor-made poly-γ-glutamic acid production.

Poly-γ-glutamic acid (γ-PGA), which is produced by several Bacillus species, is a chiral biopolymer composed of D- and L-glutamate monomers and has various industrial applications. However, synthesized γ-PGA exhibits great structural diversity, and the structure must be controlled to broaden its industrial use. The biochemical pathways for γ-PGA production suggest that the polymer properties molecular weight (MW) and stereochemical composition are influenced by (1) the affinity of γ-PGA synthetase for the two alternative glutamate enantiomers and (2) glutamate racemase activity; hence, the availability of the monomers. In this study, we report tailor-made γ-PGA synthesis with B. subtilis by combining PGA synthetase and glutamate racemase genes from several Bacillus strains. The production of structurally diverse γ-PGA was thereby achieved. Depending on the PGA synthetase and glutamate racemase origins, the synthesized γ-PGA contained 3-60% D-glutamate. The exchange of PGA synthetase changed the MW from 40 to 8500 kDa. The results demonstrate the production of low-, medium-, and high-MW γ-PGA with the same microbial chassis.

Gefitinib and curcumin-loaded nanoparticles enhance cell apoptosis in human oral cancer SAS cells in vitro and inhibit SAS cell xenografted tumor in vivo.

Curcumin (Cur), a natural product, has been shown to have anti-tumor activities in many human cancer cells. Gefitinib (Gef) is a clinical drug for cancer patients. However, there is no available information to show whether Gef/Cur nanoparticles (NPs) increased cell apoptosis and anti-tumor effects on xenograft mice model in vivo. In this study, γ-polyglutamic acid-coated nanoparticles loaded with Gef and Cur (γ-PGA-Gef/Cur NPs) were developed and its physicochemical properties and antitumor effects were investigated in vitro and in vivo. The γ-PGA-Gef/Cur NPs showed 548.5 ±â€¯93.7 nm in diameter and -40.3 ±â€¯3.87 mV on surface charge. The loading efficiencies of Gef and Cur were 89.5 and 100%, respectively. γ-PGA-Gef/Cur NPs could be internalized into SAS cells and significantly decreased total cell viability of SAS cells. Western blotting results indicated that both free Gef/Cur and γ-PGA-Gef/Cur NPs induced apoptotic cell death via caspase- and mitochondria-dependent pathways. In vivo studies indicated that treatments of PLGA NPs, free Gef/Cur, and γ-PGA-Gef/Cur NPs did not significantly affect appearances and bodyweights of mice. But the γ-PGA-Gef/Cur NPs significantly suppressed tumor size when comparing to free Gef/Cur-treated group. The nanoparticles developed in this study may be used as a potential therapy for oral cancer.

Deciphering metabolic responses of biosurfactant lichenysin on biosynthesis of poly-γ-glutamic acid.

Poly-γ-glutamic acid (γ-PGA) is an extracellularly produced biodegradable polymer, which has been widely used as agricultural fertilizer, mineral fortifier, cosmetic moisturizer, and drug carrier. This study firstly discovered that lichenysin, as a biosurfactant, showed the capability to enhance γ-PGA production in Bacillus licheniformis. The exogenous addition of lichenysin improved the γ-PGA yield up to 17.9% and 21.9%, respectively, in the native strain B. licheniformis WX-02 and the lichenysin-deficient strain B. licheniformis WX02-ΔlchAC. The capability of intracellular biosynthesis of lichenysin was positively correlated with γ-PGA production. The yield of γ-PGA increased by 25.1% in the lichenysin-enhanced strain B. licheniformis WX02-Psrflch and decreased by 12.2% in the lichenysin-deficient strain WX02-ΔlchAC. Analysis of key enzyme activities and gene expression in the TCA cycle, precursor glutamate synthesis, and γ-PGA synthesis pathway revealed that the existence of lichenysin led to increased γ-PGA via shifting the carbon flux in the TCA cycle towards glutamate and γ-PGA biosynthetic pathways, minimizing by-product formation, and facilitating the uptake of extracellular substrates and the polymerization of glutamate to γ-PGA. Insight into the mechanisms of enhanced production of γ-PGA by lichenysin would define the essential parameters involved in γ-PGA biosynthesis and provide the basis for large-scale production of γ-PGA.

Improving poly-(γ-glutamic acid) production from a glutamic acid-independent strain from inulin substrate by consolidated bioprocessing.

To excavate the application of Jerusalem artichoke on poly(γ-glutamic acid) (γ-PGA) production, a γ-PGA producing strain Bacillus amyloliquefaciens NX-2S154 was obtained through atmospheric and room temperature plasma mutagenesis, which produced 14.83 ± 0.31 g/L of γ-PGA in batch fermentation with raw inulin extract. Simultaneous saccharification and fermentation (SSF) by adding commercial inulinase were further investigated for γ-PGA fermentation. Results showed SSF could eliminate the ineffective utilization of inulin while avoiding inhibition effect of high concentration substrate, which made γ-PGA concentration reach 18.54 ± 0.39 g/L with the process being shortened by 17%. Finally, an immobilized column for reducing inulinase cost was introduced to γ-PGA production. Repeated batch cultures showed the novel bioreactor exhibited higher stability and simplicity and gave average γ-PGA concentration and productivity of 19.40 ± 0.37 g/L and 0.27 ± 0.008 g/L/h, respectively. This work proposes a productive method for efficient γ-PGA production using Jerusalem artichoke feedstock.

Vanadium: History, chemistry, interactions with α-amino acids and potential therapeutic applications.

In the last 30 years, since the discovery that vanadium is a cofactor found in certain enzymes of tunicates and possibly in mammals, different vanadium-based drugs have been developed targeting to treat different pathologies. So far, the in vitro studies of the insulin mimetic, antitumor and antiparasitic activity of certain compounds of vanadium have resulted in a great boom of its inorganic and bioinorganic chemistry. Chemical speciation studies of vanadium with amino acids under controlled conditions or, even in blood plasma, are essential for the understanding of the biotransformation of e.g. vanadium antidiabetic complexes at the physiological level, providing clues of their mechanism of action. The present article carries out a bibliographical research emphaticizing the chemical speciation of the vanadium with different amino acids and reviewing also some other important aspects such as its chemistry and therapeutical applications of several vanadium complexes.

Development of analytical methods for evaluating the quality of dissociated and associated amphiphilic poly(γ-glutamic acid) nanoparticles.

A quantitative method of analyzing nanoparticles (NPs) for drug delivery is urgently required by researchers and industry. Therefore, we developed new quantitative analytical methods for biodegradable and amphiphilic NPs consisting of polymeric γ-PGA-Phe [phenylalanine attached to poly(γ-glutamic acid)] molecules. These γ-PGA-Phe NPs were completely dissociated into separate γ-PGA-Phe molecules by adding sodium dodecyl sulfate (SDS). The dissociated NPs were chromatographically separated to analyze parameters such as the γ-PGA-Phe content in the NPs, the impurities present [using reverse-phase (RP) HPLC with an ultraviolet (UV) detector], and the absolute MW [using size-exclusion chromatography (SEC) with refractive index detection (RI) and multiangle light scattering (MALS) detection, i.e., SEC-RI/MALS]. The chromatographic patterns of the NPs were equivalent to those of the component polymer (γ-PGA-Phe), and excellent chromatographic separation for the quantitative evaluation of NPs was achieved. To the best of our knowledge, this is the first report of the quantitative evaluation of NPs in the field of NP-based delivery systems. Furthermore, these methods were applied to optimize and evaluate the NP manufacturing process. The results showed that impurities were effectively removed from the γ-PGA-Phe during the manufacturing process, so the purity of the final γ-PGA-Phe NPs was enhanced. In addition, the appearance, clarity of solution, particle size, zeta potential, particle matter, osmolarity, and pH of the product were evaluated to ensure that the NPs were of the required quality. Our approach should prove useful for product and process characterization and quality control in the manufacture of NPs. γ-PGA-Phe NPs are known to be a powerful vaccine adjuvant, so they are expected to undergo clinical development into a practical drug-delivery system. The analytical methods established in this paper should facilitate the reliable and practical quality testing of NP products, thus aiding the clinical development of γ-PGA-Phe-based drug-delivery systems. Moreover, since these analytical methods employ commonly used reagents and chromatographic systems, the methods are expected to be applicable to other NP-based drug-delivery products too. Graphical abstract NPs were completely dissociated into separate γ-PGA-Phe polymeric molecules, which yielded a similar chromatogram to that seen for the NPs.

High prevalence of Bacillus subtilis-infecting bacteriophages in soybean-based fermented foods and its detrimental effects on the process and quality of Cheonggukjang.

While the detrimental effect of bacteriophages on lactic acid bacterial fermentation is well documented, the importance of Bacillus subtilis phages in soybean-based fermented foods is not. In this study, we show for the first time that 100% of Korean soybean-based fermented foods (Doenjang, Gochujang, and Cheonggukjang) and 70% of raw materials (Meju and rice straw) were contaminated with B. subtilis-infecting phages (as high as 3.7 × 104 PFU g-1). Among 15 isolated B. subtilis-infecting phages, BSP18 was selected for further studies due to its specificity to and relatively broad host infectivity (34%) against B. subtilis. This Myoviridae family phage, BSP18 could infect all of the tested wild-type and commercially-used strains for soybean-based fermented food preparation. Furthermore, artificial contamination of as low as 102 PFU g-1 of BSP18 significantly inhibited B. subtilis growth during Cheonggukjang fermentation. Moreover, phage-treated samples contained considerably more degraded γ-PGA which could negatively affect the functional property of Cheonggukjang. We also present the data, strongly suggesting BSP18-encoded, not bacterial, γ-PGA hydrolase was responsible for γ-PGA degradation. In conclusion, B. subtilis phages are widespread in Korean soybean-based fermented foods and it should be of great concern as phages may hamper the bacterial growth during fermentation and yield poor quality products.