PGA Production Research Articles

A novel PGA/TiO2 nanocomposite prepared with poly(γ-glutamic acid) from the newly isolated Bacillus subtilis 17B strain

Poly(?-glutamic acid) (PGA), naturally produced by Bacillus species, is a biodegradable, non-toxic, biocompatible and non-immunogenic negatively charged polymer. Due to its properties, it has found various applications in the food, cosmetic and pharmaceutical industries. In this work, Bacillus subtilis 17B was selected as the best PGA producer among fifty wild-types Bacillus strains tested and characterized as a glutamate-independent producer. The production of PGA by the newly identified strain was optimized and increased tenfold using the Box?Behnken experimental design. The purity of PGA after recovery and purification from the fermentation broth was confirmed by SDS- -PAGE followed by methylene blue staining. PGA was characterized by ESI MS and used for the preparation of a new nanocomposite with TiO2. The synthesis of PGA/TiO2 nanocomposite, its structural analysis, and cytotoxic effect on the cervical cancer cell line (HeLa cell) was investigated to determine the potential anti-cancer usage of this newly prepared material. It is encouraging that PGA/TiO2 nanocomposite showed an increased cytotoxic effect compared to TiO2 alone.

Screening and characterization of potent poly glutamic acid producing Bacillus sp. isolated from Kinema, water and soil samples

Microbially produced gamma poly glutamic acid (γ–PGA) is a commercially important biopolymer with many applications in foods and various other substances and are abundantly used in different parts of the world. With an aim to study the potent γ–PGA producing Bacillus species, a total of 47 different samples (Kinema, soil, and water) were randomly collected from different locations across the country, and Bacillus sp. were selectively isolated, screened, and characterized by performing physiological, biochemical, morphological, and 16S rRNA gene sequencing. The microbial production of γ–PGA was assayed with the selected isolates on the PGA medium and the metabolite obtained was recovered by ethanol precipitation method and further characterized by thin-layer chromatography (TLC). Thermotolerance (25–60 °C), pH tolerance (4–9), and NaCl tolerance (1–9%) tests were performed to optimize the bacterial growth and γ–PGA production and its viscosity were measured by Ostwald's viscometer. Out of 145 randomly selected colonies, 63 isolates were Gram-positive, rods, and endospore producers and were presumptively confirmed as genus Bacillus. Higher growth of γ–PGA producers were reported in 22 isolates and was found at optimum conditions such as temperature (30–37 °C), pH (6.5–7), incubation time (3 days), and NaCl concentration (3%) and γ–PGA thus produced was further verified by TLC with the retention factor (RF) value 0.27. The potent isolates were closely similar to Bacillus subtilis subsp. stercoris, Bacillus cereus, Bacillus paranthracis, and Bacillus licheniformis etc. Based on the findings of the study, B. licheniformis is the most potent γ–PGA producing Bacillus sp. which can further be used for the commercial production of γ–PGA. To the best of our knowledge, there is yet no published research from Nepal showing the production of the γ–PGA although microbially produced γ–PGA are the major constituents in some popular foods in particular communities of the country.

From Residues to Added-Value Bacterial Biopolymers as Nanomaterials for Biomedical Applications.

Bacterial biopolymers are naturally occurring materials comprising a wide range of molecules with diverse chemical structures that can be produced from renewable sources following the principles of the circular economy. Over the last decades, they have gained substantial interest in the biomedical field as drug nanocarriers, implantable material coatings, and tissue-regeneration scaffolds or membranes due to their inherent biocompatibility, biodegradability into nonhazardous disintegration products, and their mechanical properties, which are similar to those of human tissues. The present review focuses upon three technologically advanced bacterial biopolymers, namely, bacterial cellulose (BC), polyhydroxyalkanoates (PHA), and γ-polyglutamic acid (PGA), as models of different carbon-backbone structures (polysaccharides, polyesters, and polyamides) produced by bacteria that are suitable for biomedical applications in nanoscale systems. This selection models evidence of the wide versatility of microorganisms to generate biopolymers by diverse metabolic strategies. We highlight the suitability for applied sustainable bioprocesses for the production of BC, PHA, and PGA based on renewable carbon sources and the singularity of each process driven by bacterial machinery. The inherent properties of each polymer can be fine-tuned by means of chemical and biotechnological approaches, such as metabolic engineering and peptide functionalization, to further expand their structural diversity and their applicability as nanomaterials in biomedicine.

Effect of pgsE expression on the molecular weight of poly(γ-glutamic acid) in fermentative production

Poly(γ-glutamic acid) (PGA) is a biopolymer produced by Bacillus spp. via the γ-amide linkages of d- and/or l-glutamate. Although high-molecular-weight (HMW) PGA possesses many attractive properties, such as flocculating, wound healing, and immune-stimulating effects, no studies have reported factors useful for increasing the molecular weight of PGA during microbial production. PgsB, PgsC, and PgsA are the minimum protein sets required for PGA production in B. subtilis, and PgsE improves PGA productivity. Analysis by size-exclusion chromatography combined with multiangle laser light scattering revealed that the molecular weight of PGA was Mw = 2,900,000 g mol−1 and predominantly Mw = 47,000 g mol−1 in preparations derived from B. subtilis cells with and without pgsE, respectively. PgsE may be required to increase the molecular weight of PGA. Poly(γ-glutamic acid) (PGA) is a biopolymer produced by Bacillus spp. via the γ-amide linkages of d- and/or l-glutamate. PgsB, PgsC, and PgsA are the minimum protein set required for PGA production in B. subtilis, and PgsE improves PGA productivity. Analysis by size-exclusion chromatography combined with multiangle laser light scattering revealed that the molecular weight of PGA was Mw = 2,900,000 g mol−1 or predominantly Mw = 47,000 g mol−1 in preparations derived from B. subtilis cells with or without pgsE, respectively. PgsE may be required to increase the apparent molecular weight of PGA.

A review on synthesis and biomedical applications of polyglycolic acid

Poly(glycolic acid) (PGA) is an essential biopolymer due to its thermal and mechanical properties and biodegradability which provide utility for medical applications and renewable industry. For biomedical applications, production of PGA with high molecular weight is an essential factor to possess adequate mechanical stability. Primary pathways for PGA synthesis are ring-opening polymerization of glycolide (ROP), direct polycondensation of glycolic acid, and solid-state polycondensation of halogen acetates. For PGA synthesis, different systems have been developed with using varying parameters including catalysis, initiators, solvents, and reaction temperature. This review summarizes the different synthesis pathways and physicochemical properties of PGA. Biomedical applications of PGA are also discussed.

Optimization of Some Nutritional Conditions and α- Ketoglutaric Acid Concentration as PGA Precursor for Maximizing PGA Production from Bacillus sp. 42 and Bacillus sonorensis 44

Poly gamma glutamic acid is a biodegradable, water soluble and non-toxic edible biopolymer, PGA has nylon back bone similar structure and expressed as bio-nylon. Various bacterial strains produced PGA on of them Bacillus sp. such as B. subtilis, B. lichanformans and B. sonorensis. Polymer yield was affected with medium composition as nitrogen and carbon sources. The current experimental was carried out using shake flask technique for PGA production during 72 of fermentation. The highest biomass was achieved at glycerol media and glucose media for PGA yield and productivity being 2.31, 9.65 gl-1 and 0.134 gl-1h-1, respectively of B. sonorensis 44. Of nitrogen source, organic source (yeast extract) was higher PGA yield and productivity than inorganic sources (NH4NO3) which reduced PGA yield about 28.7 and 36.02% of Bacillus sp. 42 and B. sonorensis 44, respectively. Production media supple- mented with 0.5 and 0.75 gl-1 α-keto-glutaric acid increased PGA yield about 1.24fold for both Bacillus strains. Osmotic pressure of 2.55 MPa (3% NaCl) enhanced PGA yield about 1.18 and 1.24fold of Bacillus sp. 42 and B. sonorensis 44, respectively. Furthermore, the highest PGA was received using medium containing glucose and yeast extract (as C and N2 sources). α-keto-glutaric acid and osmotic potential has an induction effect for polymer accumulation.

Isolation, Identification and Evaluation of Egyptian Bacillus sp. Isolates for Producing Poly-γ-glutamic Acid

Poly-γ-glutamic acid (γ-PGA) is a non-toxic, biodegradable, and water-soluble polymer secreted from different microbes. We isolated forty-nine Bacillus spp. isolates generating PGA from the soil of different Egyptian regions. A total of 18% of these isolates were found to highly produce PGA in the range of 5- 8 gl-l. Two of them, B44 and B42, were identified by the16SrRNA DNA sequence, and they shared 81% of similarity. The most similar DNA sequences to B44-16SrRNA were Bacillus sp. (FJ607057.1) and Bacillus sonorensis (KP236346.1) matching 99% and 94% of similarity, respectively. However, B42-16SrRNA was similar 99% to several Bacillus species. The tree-building algorithm MEGA-X constructed the phylogenetic tree of 16SrRNA DNA sequences for Bacillus 44 and Bacillus 42 strains along with other similar Bacillus species revealing the distance between them. We also boosted the PGA production of both strains. Bacillus 44 strain revealed the highest accumulation level of PGA at 35°C and 72 h of incubation using medium M with inoculum size and agitation speed 3% and 250 rpm, respectively. Both strains secreted the PGA biopolymer with a molecular mass of 55 kDa. This investigation is an attempt of boosting promising Egyptian Bacillus sp. isolates for PGA production that may be a seed for industrial production.

Production and physicochemical characterization of bacterial poly gamma- (glutamic acid) to investigate its performance on enhanced oil recovery

Bacillus licheniformis LMG 7559, which is capable of producing extracellular poly gamma- (glutamic acid) (PGA), was provided for the biopolymer synthesis. Using a modified PGA medium for PGA production, the isolated biopolymer, undergone dialysis process mainly for desalination and removal of other impurities. The bacteria produced high molecular weight biopolymers with a weight average molecular weight (M̅n) of 1.6 × 105 g/mole identified by gel permeation chromatography (GPC). Furthermore, GPC analysis was utilized to determine the poly-dispersity of PGA as well as molecular weight variation by cultivation time. The heavy weight fraction of 1.85 × 105 g/mole with poly-dispersity index of 7.42 was distinguished. For the extracted and dialyzed biopolymer, thermal properties were studied using DSC/TGA by which a mass loss of 36 percent was observed. Eventually, the biopolymer solution was injected into the oil saturated heterogeneous porous medium to evaluate the recovery factor enhancement by PGA flooding. It was found that 31.45% of oil in place was recovered by biopolymer flooding, whereas only 16.6% of oil in place was obtained by water flooding.

A versatile method for the synthesis of poly(glycolic acid): high solubility and tunable molecular weights

Poly(glycolic acid) (PGA) is an important biopolymer, especially in medical applications because of its suitable mechanical, biocompatible, and biodegradable properties. PGA can degrade within weeks, depending on its molecular weight. Production of high molecular weight PGA is important to achieve sufficient mechanical stability for biomedical applications. High molecular weight PGA is difficult to obtain by direct condensation of the related carboxylic acids; therefore, polyglycolide is typically made by ring opening polymerization of the cyclic diester glycolide. However, this procedure is restrictive because of the high cost of the raw material (glycolide) and the associated high energy consumption. Here, we describe the synthesis of PGA via an azeotropic distillation method that enables tunable molecular weights. The synthesized PGA is highly soluble in organic solvents and degrades faster than reference PGA. We described the synthesis of PGA via an azeotropic distillation method that enables tunable molecular weights. This alternative method produces a white powdered PGA rather than a waxy brown product that is obtained by conventional melt-solid condensation methods. Additionally, improved solubility renders this method useful for producing soluble high molecular weight PGA for further applications. Highly crystalline PGA with a high molecular weight (Mn = 32,100 g/mol) was obtained with a homogenous polydispersity index of 1.2–1.4.

Lack of the PGA exopolysaccharide in Salmonella as an adaptive trait for survival in the host.

Many bacteria build biofilm matrices using a conserved exopolysaccharide named PGA or PNAG (poly-β-1,6-N-acetyl-D-glucosamine). Interestingly, while E. coli and other members of the family Enterobacteriaceae encode the pgaABCD operon responsible for PGA synthesis, Salmonella lacks it. The evolutionary force driving this difference remains to be determined. Here, we report that Salmonella lost the pgaABCD operon after the divergence of Salmonella and Citrobacter clades, and previous to the diversification of the currently sequenced Salmonella strains. Reconstitution of the PGA machinery endows Salmonella with the capacity to produce PGA in a cyclic dimeric GMP (c-di-GMP) dependent manner. Outside the host, the PGA polysaccharide does not seem to provide any significant benefit to Salmonella: resistance against chlorine treatment, ultraviolet light irradiation, heavy metal stress and phage infection remained the same as in a strain producing cellulose, the main biofilm exopolysaccharide naturally produced by Salmonella. In contrast, PGA production proved to be deleterious to Salmonella survival inside the host, since it increased susceptibility to bile salts and oxidative stress, and hindered the capacity of S. Enteritidis to survive inside macrophages and to colonize extraintestinal organs, including the gallbladder. Altogether, our observations indicate that PGA is an antivirulence factor whose loss may have been a necessary event during Salmonella speciation to permit survival inside the host.

Metabolome analysis reveals the effect of carbon catabolite control on the poly(γ-glutamic acid) biosynthesis of Bacillus licheniformis ATCC 9945

Poly(γ-glutamic acid) (PGA) is a polymer composed of L- and/or D-glutamic acids that is produced by Bacillus sp. Because the polymer has various features as water soluble, edible, non-toxic and so on, it has attracted attention as a candidate for many applications such as foods, cosmetics and so on. However, although it is well known that the intracellular metabolism of Bacillus sp. is mainly regulated by catabolite control, the effect of the catabolite control on the PGA producing Bacillus sp. is largely unknown. This study is the first report of metabolome analysis on the PGA producing Bacillus sp. that reveals the effect of carbon catabolite control on the metabolism of PGA producing Bacillus licheniformis ATCC 9945. Results showed that the cells cultivated in glycerol-containing medium showed higher PGA production than the cells in glucose-containing medium. Furthermore, metabolome analysis revealed that the activators of CcpA and CodY, global regulatory proteins of the intracellular metabolism, accumulated in the cells cultivated in glycerol-containing and glucose-containing medium, respectively, with CodY apparently inhibiting PGA production. Moreover, the cells seemed to produce glutamate from citrate and ammonium using glutamine synthetase/glutamate synthase. Pulsed addition of di-ammonium hydrogen citrate, as suggested by the metabolome result, was able to achieve the highest value so far for PGA production in B. licheniformis.

Efficient production of poly‐γ‐glutamic acid from cane molasses by Bacillus subtilisNX‐2 immobilized on chemically modified sugarcane bagasse

AbstractBACKGROUNDPoly‐γ‐glutamic acid (PGA), a naturally occurring homopolymer, has gained increased interest worldwide because of its extensive applications. In this study, sugarcane bagasse chemically modified with poly‐ϵ‐L‐lysine (PL) was used in an aerobic plant fibrous‐bed bioreactor (APFB) to enhance PGA fermentation by Bacillus subtilis NX‐2.RESULTS5 g L−1 of PL was optimized to modify native sugarcane bagasse, and 45.7 ± 0.6 g L−1 PGA was obtained with PL‐bagasse immobilized cells in batch fermentation, with a productivity of 1.08 ± 0.02 g L−1 h−1. These results were 1.23 and 1.4 times higher than those with unmodified sugarcane bagasse. The effect of dissolved oxygen (DO) on PGA fermentation was further investigated, and suitable conditions were 45% DO with the PGA concentration 55.4 ± 0.4 g L−1. In 10 batches of repeated‐batch fermentation, PGA production significantly increased and the average PGA yield was maintained at 90.6 ± 0.5 g L−1 in the three later batches and was increased by 8.7% compared with that in the first batch.CONCLUSIONThe improved APFB system was proved to be a green, economic, and effective bioprocess for PGA fermentation, and this system could also provide a suitable microenvironment for B. subtilis NX‐2 to induce its evolution. © 2015 Society of Chemical Industry

Highly efficient rice straw utilization for poly-(γ-glutamic acid) production by Bacillus subtilis NX-2

Lignocellulosic biomass has been identified as an economic and environmental feedstock for future biotechnological production. Here, for the first time, poly-(γ-glutamic acid) (PGA) production by Bacillus subtilis NX-2 using rice straw is investigated. Based on two-stage hydrolysis and characteristic consumption of xylose and glucose by B. subtilis NX-2, a co-fermentation strategy was designed to better accumulate PGA in a 7.5L fermentor by two feeding methods. The maximum cumulative respective PGA production and PGA productivity were 73.0±0.5gL−1 and 0.81gL−1h−1 by the continuous feeding method, with carbon source cost was saved by 84.2% and 42.5% compared with glucose and cane molasse, respectively. These results suggest that rice straw, a type of abundant, low-cost, non-food lignocellulosic feedstock, may be feasibly and efficiently utilized for industrial-scale production of PGA.

High-throughput strategies for penicillin G acylase production in rE. colifed-batch cultivations

BackgroundPenicillin G acylase (PGA) is used industrially to catalyze the hydrolysis of penicillin G to obtain 6-aminopenicillanic acid. In Escherichia coli, the most-studied microorganism for PGA production, this enzyme accumulates in the periplasmic cell space, and temperature plays an important role in the correct synthesis of its subunits.ResultsThis work investigates the influence of medium composition, cultivation strategy, and temperature on PGA production by recombinant E. coli cells. Shake flask cultures carried out using induction temperatures ranging from 18 to 28°C revealed that the specific enzyme activity achieved at 20°C (3000 IU gDCW-1) was 6-fold higher than the value obtained at 28°C. Auto-induction and high cell density fed-batch bioreactor cultures were performed using the selected induction temperature, with both defined and complex media, and IPTG and lactose as inducers. Final biomass concentrations of 100 and 120 gDCW L-1, and maximum enzyme productivities of 7800 and 5556 IU L-1 h-1, were achieved for high cell density cultures using complex and defined media, respectively.ConclusionsTo the best of our knowledge, the volumetric enzyme activity and productivity values achieved using the complex medium are the highest ever reported for PGA production using E. coli. Overall PGA recovery yields of 64 and 72% after purification were achieved for crude extracts obtained from cells cultivated in defined and complex media, respectively. The complex medium was the most cost-effective for PGA production, and could be used in both high cell density and straightforward auto-induction protocols.

Enhanced poly(γ-glutamic acid) fermentation by Bacillus subtilis NX-2 immobilized in an aerobic plant fibrous-bed bioreactor

To enhance poly(γ-glutamic acid) (PGA) production, a novel aerobic plant fibrous-bed bioreactor (APFB) was constructed for immobilized fermentation. Based on the analysis of the kinetics of immobilized-cell fermentation using the APFB and conventional free-cell fermentation, immobilized-cell fermentation exhibited more efficient PGA production. Furthermore, repeated fed-batch cultures for PGA production were conducted to evaluate the stability of the APFB system. Average final PGA concentration and productivity of 71.21±0.83g/L and 1.246±0.008g/L/h were respectively achieved by cells immobilized in bagasse during APFB, which was reused eight times over a period of 457±18h. Analysis of the membrane phospholipids and the key enzyme activities indicated that APFB-adapted cells had better productivity than original cells. Thus, this study demonstrated the significant potential of the APFB culture system in future industrial applications.

청국장에서 분리한 Bacillus subtilis GS-2에 의한 Poly(γ-Glutamic Acid) 생산의 최적 배양조건

ABSTRACT A bacterium strain GS-2 isolated from the Korean traditional seasoning food, Chungkookjang and was determined to produce large amounts of γ-PGA with high productivity when provided with simple nutrients (L-glutamic acid 2.0%, glucose 1.0%, NH 4 Cl 0.5%, KH 2 PO 4 0.05%, MgSO 4 ․ 7H 2 O 0.01%, pH 7.0). In this study, the culture medium for this strain was optimized for the production of γ-PGA. The Bacillus subtilis GS-2 required supplementation with L-glutamic acid and other nutrients for maximal production of γ-PGA. The optimal culture conditions for γ-PGA production were a 48 hr culture time, a temperature of 33℃ and initial pH of 6.5 by rotary shaking (220 rpm). A maximum γ-PGA production of 31.0 g/l was obtained with L-glutamic acid (30 g/ l ), sucrose (the main carbon source, 30 g/l), NH 4 Cl (the main nitrogen source, 2.5 g/l), KH 2 PO 4 (1.5 g/l) and MgSO 4 ․ 7H 2 O (0.15 g/l) in the culture medium. REFERENCES Ashiuchi M, Kamei T, Baek DH, Shin SY, Sung MH, Soda K, Yagi T, Misono H. 2001. Isolation of Bacillus subtilis (Chungkookjang), a poly-γ-glutamate producer with high genetic competence. Appl Microbiol Biotechnol 57:764-769Back LM, Park LY, Park KS, Lee SH. 2008. Effect of starter cultures on the fermentative characteristics of Cheonggukjang. Kor J Food Sci Technol 40:400-405Bang BH, Jeong EJ, Rhee MS, Kim YM, Yi DH. 2011. Isolation of Bacillus subtilis GS-2 producing γ-PGA from Chungkukjang bean paste and identification of γ-PGA. J Appl Biol Chem 54:1-6Borbely M, Nagasaki Y, Borbely J, Fan K, Bhogle A, Sevoian M. 1994. Biosynthesis and chemical modification of poly (-γ-glutamic acid). Polymer Bull 32:127-132Bovarnick M. 1942. The formation of extracellular D(-)glutamic acid polypeptide by Bacillus subtilis. J Bio Chem 145:415- 424Bradford MM. 1976. A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254Cheng C, Asada Y, Asada T. 1989. Production of γ-poly- glutamic acid by Bacillus subtilis A 35 under denitrifynig conditions. Agric Biol Chem 53:2369-2375Choi HJ, Yang R, Kunioka M. 1995. Synthesis and charac-terization of pH-sensitive and biodegradable hydrogels pre-pared by γ-irradiation using microbial poly(γ-glutamic acid) and poly(e- lysine). J Appl Polym Sci 58:807-814Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. 1956. Colorimetric method for determination of sugar and related substances. Anal Che 28:350-356Francis F, Sabu A, Madhavan Nampoothiri K, Ramachandran S, Ghosh S, Szakacs G, Pandey A. 2003. Use of response surface methodology for optimizing process parameters for the production of α-amylase by Aspergillus oryzae. Biochem Eng J 15:107-115Goto A, Kunioka M. 1992. Biosynthesis and hydrolysis of poly (-γ-glutamic acid) from Bacillus subtilis IFO 3335. Biosci Biotechnol Biochem 56:1031-1035Hara T, Ueda S. 1982. Regulation of polyglutamate production in Bacillus subtilis(natto); transformation of high PGA pro-ductivity. Agric Biol Chem 46: 2275-2281Hasebe K, Inagaki M. 1999. Preparation composition for external use containing gamma-polyglutamic acid an vegetable extract in combination. JP. Patent 11240827Ito Y, Tanaka T, Asada Y. 1996. Glutamic acid independent pro-duction of poly(-γ-glutamic acid) by Bacillus subtilis TAM- 4. Biosci Biotechnol Biochem 60:1239-1242Kambourova M, Tangney M, Priest FG. 2001. Regulation of poly glutamic acid synthesis by glutamate in Bacillus licheniformis and Bacillus subtilis. Appl Environ Microbiol 67:1004-1007Kubota H, Matsunobu T, Uotani K, Takebe H, Satoh A, Tanaka T, Tanguchi M. 1993. Production of poly(γ-glutamic acid) by Bacillus subtilis F-2-01. Biosci Biotechnol Biochem 57: 1212-1213Leonard CG, Housewright RD, Thorne CR. 1958. Effects of some metallic ions on glutamyl polypeptide synthesis by Bacillus subtilis. J Bacteriol 76:499-503Markland P, Amidon GL, Yang VC. 1999. Modified polypeptides

Effects of oxygen vectors on the synthesis and molecular weight of poly(γ-glutamic acid) and the metabolic characterization of Bacillus subtilis NX-2

The effects of different oxygen vectors on the synthesis and molecular weight of poly(γ-glutamic acid) (PGA) were investigated in the batch fermentation of Bacillus subtilis NX-2. n-Hexane, n-heptane, and n-hexadecane enhanced the PGA concentration and molecular weight. The PGA concentration reached a maximum of 39.4±0.19gL−1, and the highest molecular weight obtained was (19.0±0.02)×105Da with the addition of 0.3% n-heptane. However, n-dodecane decreased the PGA concentration and molecular weight to final values of 20.1±0.10gL−1 and (8.4±0.02)×105Da, respectively. Analysis of the intracellular nucleotide levels of B. subtilis NX-2 with n-heptane and n-dodecane additives showed that the lowest NADH/NAD+ ratio and ATP levels were obtained with the n-dodecane additives, which can explain the decreased PGA yield and molecular weight. The metabolic flux distribution of B. subtilis NX-2 with n-heptane and n-dodecane additives was also investigated. Flux distribution was primarily directed to the EMP and TCA cycles with n-heptane additives. The flux of 2-oxoglutarate to intracellular glutamate and the flux distribution from extracellular to intracellular glutamate both increased to improve PGA production.

Economical production of poly(γ-glutamic acid) using untreated cane molasses and monosodium glutamate waste liquor by Bacillus subtilis NX-2

The production of poly(γ-glutamic acid) by Bacillus subtilis NX-2 from cane molasses and monosodium glutamate waste liquor (MGWL) was studied for the first time in this work. When batch fermentation was carried out with untreated molasses, 33.6±0.37gL−1 PGA was obtained with a productivity of 0.46±0.006gL−1h−1. In order to minimize the substrate inhibition, fed-batch fermentation was performed with untreated or hydrolyzed molasses in 7.5L bioreactor, giving 50.2±0.53 and 51.1±0.51gL−1 of PGA at 96h, respectively. Further studies were carried out by using MGWL as another carbon source, resulting in a PGA concentration of 52.1±0.52gL−1 with a productivity of 0.54±0.003gL−1h−1. These results suggest that the low-cost cane molasses and MGWL can be used for the environmental-friendly and economical production of PGA by B. subtilis NX-2.

Improvement of poly(γ-glutamic acid) biosynthesis and quantitative metabolic flux analysis of a two-stage strategy for agitation speed control in the culture of Bacillus subtilis NX-2

In this study, the production of poly(γ-glutamic acid) by Bacillus subtilis NX-2 (PGA) at different agitation speeds was investigated. Based on the analysis of specific cell growth rate (μ) and specific PGA formation rate (qp), a two-stage strategy for agitation speed control was proposed. During the first 24 h, an agitation speed of 600 rpm was used to maintain a high μ for better cell growth, which then reduced to 400 rpm after 24 h to maintain a high qp to enhance PGA production. Using this method, the maximum concentration of PGA reached 40.5 ± 0.91 g/L and the PGA productivity was 0.56 ± 0.012 g/L/h, which was 17.7 and 9.8% higher, respectively, than the best results obtained when a constant agitation speed was used. The flux distributions and the related enzymes of 2-oxoglutarate could be affected by this two-stage strategy for agitation speed. The activity of isocitrate dehydrogenase and glutamate dehydrogenase at the key node of 2-oxoglutarate increased, and more flux distribution was directed to glutamate. The flux distribution from extracellular to intracellular glutamate also increased and improved PGA production as the glutamate uptake rates increased using the agitation-shift control method.

A facile pretreatment method for efficient immobilization of penicillin G acylase

A facile pretreatment method was developed to obtain high specific activity of PGA from recombinant E. coli cells for preparing immobilized enzyme with high activity, which coupled selective extraction of PGA with butyl acetate and adsorption of butyl acetate with active carbon. Butyl acetate (5%, v/v) led to a 92.0% release of PGA, the specific activity of which in the extracting solution was twice that obtained by sonication. The negative effect on PGA immobilization due to residual butyl acetate in the extracting solution was removed by adding active carbon (8%, w/v). The final PGA solution meets requirement for industrial immobilization while eliminating the high cost of a traditional purification process. This pretreatment method developed in this work is simple, highly performing and cost-effective. It has been employed in a 10 tons/year immobilized PGA production line.