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Optimization of Culture Conditions for Production of Polyamide Biopolymer (Polyglutamate) by Bacillus sp. Strain-R
Abstract
An optimization study of biopolymer production by locally isolated Bacillus sp. strain-R was carried out, where analysis indicated that it is a polyamide homopolymer consists mainly of glutamate. Preliminary experiment to address the most suitable carbon and nitrogen sources revealed that a production of 14.25 g L<sup>-1</SUP> of the polyamide (polyglutamate) was achieved in the presence of glucose and ammonium sulfate. To evaluate the effect of different culture conditions on the production of PGA, Plackett-Burman factorial design was carried out. Ten variables were examined for their significance on PGA production. Among those variables K<SUB>2</SUB>HPO<SUB>4</SUB>, KH<SUB>2</SUB>PO<SUB>4</SUB>, (NH<SUB>4</SUB>)<SUB>2</SUB>SO<SUB>4 </SUB>and glucose were found to be the most significant variables that encourage PGA production. The pre-optimized medium showed approximately three folds increase in PGA production.
... Bacillus subtilis and Bacillus licheniformis are the most conspicuous bacteria producing PGA (Ko and Gross, 1998;Yoon et al., 2000;Ashiuchi et al., 2001;Hoppensack et al., 2003;Soliman et al., 2005;Berekaa et al., 2006). In B. anthracis and B. megaterium, PGA exists as a capsular exocellular polymer and serves as structural component (Hanby and Rydon, 1946;Roelants and Goodman, 1968). ...
... There is a general practice of determining optimal concentration of media components by varying one-variable-at-a-time (OVAT). However, experimental design techniques present more balanced alternative to one-variable-at-a-time (OVAT) approach to fermentation improvement in PGA production (Soliman et al., 2005;Berekaa et al., 2006). In this study, PGA-producing Bacillus sp. ...
... The plates were dried and sprayed with acetone containing 0.2% Ninhydrin to visualize the amino acid (Kambourova et al., 2001). On the other hand, the precipitated polymer material was hydrolyzed at 110°C for 24 h in a sealed evacuated tube, and amino acid composition was determined with Beckman system analyzer (Kambourova et al., 2001;Berekaa et al., 2006). Plackett-Burman experimental design (1946) was applied to investigate the significance of various medium components on PGA production. ...
... Bacterial isolates were activated in N.B for 18hrs, 2ml of this culture was added to 100ml of glutaminase liquid medium [8] and incubated at 37°C for 24hrs In shaking incubation at 100rpm. ...
Five Bacillus sp were obtained from soil, one isolate was selected according its, highest enzyme productivity, it was identified as Bacillus BG. The bacteria was cultured in a liquid medium, the enzyme precipitated by 30% saturation of ammonium sulphate, dialyzed and immobilized by adsorption on different supports including sephadexG-25, DEAE-cellulose, chitin powder, sawdust, charcoal. DEAE-cellulose retained most of enzyme activity (90%) followed by chitin (78%), then sephadex G-25 (73%) while sawdust and charcoal retained (55%) only. The immobilized enzyme was subjected to different temperatures charcoal was the best matrix for protecting glutaminase against heat, it retained 55 and 22% of original activity chitin after 2hrs of incubation at 50 and 60°C respectively while the free enzyme retained 30 and 10% at the same condition. The immobilized enzyme was more stable at pH 8 than at pH 5. The enzyme adsorbed on DEAE-cellulose retained the maximum activity (98%) at pH 8 for 2 hrs, while it was 73% for the free enzyme. It can be concluded from these results that Bacillus subtilis glutaminase can be immobilized on different inert materials
... Bacterial isolates were activated in N.B for 18hrs, 2ml of this culture was added to 100ml of glutaminase liquid medium [8] and incubated at 37°C for 24hrs In shaking incubation at 100rpm. ...
... Medium optimization by application of statistical optimization method, compared to the common "one-factor-at-a-time" method, proved to be powerful and useful tool. Recently, application of statistical methods and response surface methodology (RSM) has gained a lot of impetus for medium optimization and understanding the interactions among various physicochemical parameters involved in biopolymer production (Khanna and Srivastava, 2005;Nikel et al., 2005;Berekaa et al., 2006;Sharma et al., 2007;Yu et al., 2008;Mu et al., 2009;Mokhtari-Hosseini et al., 2009;Pandian et al., 2010). ...
Bacillus megaterium SW1-2 producing polyhydroxalkanoate (PHA), previously isolated from activated sewage sludge was selected for the study of PHB biopolymer production. Preliminary experiment showed that the strain capable of producing 36% cell dry weight (CDW) of the polymer during growth on basal E2 medium supplemented with glucose, followed by 28, 15 and 8% CDW during growth on Na-acetate, lactose or Na-succinate, respectively. Spectroscopic analysis by FT-IR revealed characteristic peaks at 1722.8 and 1276 cm -1 corresponding to the C=O and C-O stretching groups indicated that the polymer composed mainly of polyhydroxybutyrate (PHB). Statistically based experiments were applied to optimize the culture conditions for production of PHB. Four selected parameters namely; ammonium sulfate, glucose, KH 2 PO4 and Na 2 HPO 4 , respectively, were further investigated using Box-Behnken design to define the optimal production condition. Based on statistical analysis, maximal PHB production (1.45 g/L) was reached using optimal medium composition, which is more than 1.8-folds the basal medium. Verification experiment was carried out to examine model validation and revealed more than 75% validity.
... However, not many studies are available on the fungi Aspergillus nidulans to exploit its potential in the production of cellulase. In the present study, one factor at a time methodology (OFAT) (Skowronek & Fiedurek, 2004), Plackett Burmann methodology (PB) (Berekaa et al., 2006) and response surface methodology (RSM) (Amani et al., 2007) have been adopted to optimize the medium requirements for the production of cellulase by A. nidulans. The central composite design (Guangrong et al., 2008) was used to optimize the levels of identified controllable factors affecting the medium. ...
The enhancement of the cellulase activity of Aspergillus nidulans by combinational optimization technique was investigated. The strain isolated from decayed, dry leaf of Ficus caricus was compared for the first time for its ability to produce cellulolytic enzyme in submerged fermentation (SmF). The medium ingredients enhancing the cellulase production were optimized by combinational statistical approach by one factor at a time methodology (OFAT), Plackett Burmann methodology (PB) and response surface methodology (RSM). A four–factor–five-level central composite design (CCD) was employed to determine the maximum activity of cellulase at optimum levels of carboxy methyl cellulose (CMC), ammonium nitrate and potassium dihydrogen phosphate at varying pH values. The optimum fermentation parameters were found to be 1.2 g/l CMC, 0.9 mg/l ammonium nitrate and 0.75 mg/l potassium dihydrogen phosphate at pH 6. The optimization of medium by combinational statistical approach led to the fine tuning of the cellulase production thereby enhancing the cellulase activity from 4.91 U/ml to 39.56 U/ml. The predicted results were in agreement with the actual experimental values. The cellulase activity obtained with this strain may be one of the best obtained in Aspergillus nidulans.
... The Bacillus strain used throughout this study was isolated from an agriculture farm in ABIS rectorate in Egypt, characterized by previous history of fertilizer's use, as previously described (6). Center, Azhar University revealed that the bacterium was closely related to Bacillus licheniformis. ...
Production of Polyglutamate (PGA) biopolymer by immobilized Bacillus licheniformis strain-R was intensively investigated. Preliminary experiments were carried out to address the most suitable immobilization methodology. Entrapment of Bacillus cells in alginate-agar led optimal PGA production (36.75 g/l), with 1.32-and 2.18-fold increase in comparison with alginate-or K-carrageenan-immobilized cells, respectively. During semicontinuous cultivation of agar-alginate gel-cell mixture, production of PGA by 10 ml mixture was increased from 2(nd) to 3(rd) run whereas, increased till the 4(th) run using 15ml mixture. Adsorption was the most suitable immobilization technique for production of PGA and the sponge cubes was the preferred matrix recording 43.2 g/l of PGA with the highest cell adsorption. Furthermore, no PGA was detected when B. licheniformis cells were adsorbed on wood and pumice. Although luffa pulp-adsorbed cells recorded the highest PGA production (50.4 g/l), cell adsorption was the lowest. Semicontinuous cultivation of B. licheniformis cells adsorbed on sponge led to increase of PGA production till the 3(rd) run and reached 55.5 g/l then slightly decreased in the 4(th) run. The successful use of fixed-bed bioreactor for semicontinuous cultivation of B. licheniformis cells held on sponge cubes (3 runs, 96 hours/run) provides insight for the potential biotechnological production of PGA by immobilized cells.
Polyglutamic acid (PGA), a protein in the mucilage of PGA-producing Bacillus spp., has expected applications in medical and biotechnological industries. Although the Bacillaceae family contains over 100 genera, research on bacterial PGA has exclusively focused on the genus Bacillus, especially B. subtilis var. natto and B. licheniformis. In the present study, indigenous Bacillaceae family strains were isolated from withered leaves and soil samples and screened for PGA production. As a result of the screening, the strain 8h was found to produce a mucilage possessing greater viscosity than PGA of B. subtilis var. natto (natto PGA). Biochemical analyses revealed that the 8h mucilage contains 63% protein and 37% polysaccharide, while mucilage of B. subtilis var. natto is composed of 61% protein and 39% polysaccharide. The most plentiful amino acid in 8h mucilage protein was glutamate (43%, mol/mol), which is similar to that of natto PGA, suggesting that it possesses characteristics of PGA. Although natto mucilage contains fructan, glucan was found as the polysaccharide of 8h mucilage. While phylogenetic studies indicated that the strain 8h belongs to Peribacillus simplex, the yield of the viscous mucilage by strain 8h was significantly higher than P. simplex type strain, suggesting that 8h is a mucilage-overproducing strain of P. simplex. Interestingly, 8h mucilage protein was found to contain more hydrophobic amino acid residues than natto PGA, suggesting that its amphiphilicity is suitable as a drug carrier and adjuvant. The present study is the first report of viscous mucilage and PGA-like protein produced by the genus Peribacillus.
The present study employs the use of combinatorial statistical approach for the optimization of media components for the enhanced production of complete cellulase system by Aspergillus niger C-5. Kitchen waste residue, a biodegradable fraction of municipal solid waste was used as a substrate for growth and production of complete fungal cellulase system under solid state fermentation conditions. A total of 23 media components affecting cellulase yields were first statistically screened employing a 2-level Plackett-Burman design model. Response surface methodology (RSM) was then used to optimize the concentration of most significant factors including tryptone, SDS, NH4Cl and NaCl affecting cellulase yields. The concentrations of four medium components that led to the maximum production of complete cellulase system as optimized using RSM were tryptone, 400 mg; SDS, 0.60 mg; NH4Cl, 2.5 mg; NaCl 1.50 mg/5 g of dry substrate. The optimization of medium components by combinatorial statistical approach led to the fine tuning of cellulase production thereby enhancing the activities of all components viz CMCase, FPase and β-glucosidase by 2.04, 1.58, 1.74 folds respectively exhibiting the highest yields of 110, 19 and 66 U/g dry substrate.
Poly(γ-d-glutamic acid) (PGA)-producing strains ofBacillus species were investigated to determine their ability to contribute to reducing the amount of ammonium nitrogen in liquid
manures and their ability to convert some of the ammonium into this polyamino acid as a transient depot for nitrogen. Organisms
that do these things should help solve the serious environmental problems which are caused by the use of large amounts of
liquid manure resulting from intensified agriculture; these problems are mainly due to the high content of ammonium nitrogen.
Bacillus licheniformis ATCC 9945 and Bacillus subtilis were able to grow in liquid manure and to produce PGA in the presence of sodium gluconate. On artificial liquid manure these
two strains were able to produce 0.85 and 0.79 g of PGA per liter, respectively. Under conditions that are found in intensified
farming situations the ammonia content was reduced within 48 h from 1.3 to 0.75 g/liter. One mutant of B. subtilis 1551 impaired in the catabolism of PGA was obtained after nitrosoguanidine mutagenesis. This mutant produced PGA at a final
concentration of 4.8 g/liter, whereas the wild type produced only 3.7 g/liter.
γ-Poly(glutamic acid), γ-PGA, is a bacterially synthesized water soluble nylon. It can be classified as a pseudo-poly(amino acid) which contains only glutamate repeat units. γ-PGA differs from proteins, however, in that the glutamate repeat units are polymerized by a ribosome-independent process. Furthermore, the glutamate repeat units are linked between the α-amino and γ-carboxylic acid functional groups (see below) [1].
Two isozymes of gamma-glutamyltranspeptidase, GGT-A and GGT-B, were purified to electrophoretic homogeneity from a culture broth of Bacillus subtilis TAM-4, which produces poly(gamma-glutamic acid) (PGA) de novo. GGT-A was composed of three subunits with molecular weights of 23,000 (I), 39,000 (II), and 40,000 (III). GGT-B was composed of two subunits with molecular weight of 22,000 (I) and 39,000 (II). The N-terminal amino acid sequences of GGT-A subunit I and GGT-B subunit I were very similar. GGT-A subunit II and GGT-B subunit II had an identical N-terminal amino acid sequence. That of GGT-A subunit III showed no similarity to the other subunits. Both GGTs had similar enzymatic properties (optimum pH and temperature: pH 8.8 and 55 degrees C) but showed a significantly different thermal stability at 55 degrees C. Both GGT-A and -B used D-gamma-glutamyl-p-nitroanilide as well as the L-isomer as the gamma-glutamyl donor and used various amino acids and peptides as the acceptor. It was also found that the PGA produced by the strain was hydrolyzed to glutamic acid by its own GGTs.
A bacterium that produced a large amount of poly(γ-glutamic acid) (PGA) when it was grown aerobically in a culture medium containing ammonium salt and sugar as sources of nitrogen and carbon, respectively, was isolated from soil. The bacterium, strain TAM-4, was classified as Bacillus subtilis. The maximum PGA production (22.1 mg/ml) was obtained when it was grown in a medium containing 1.8% ammonium chloride and 7.5% fructose at 30°C for 96 h with shaking. Some properties of the PGA obtained at different times of cultivation were investigated by gel permeation chromatography, SDS-PAGE, and measurement of viscosity, and calculation of the D/L ratio of glutamic acid constituting PGA. The results suggested that PGA was elongated with no changes in the diastereoisomer ratio in the molecule.
Poly-γ-o-glutamic acid has not been found to be immunogenic in pure form but elicits antipolypeptide antibodies when complexed with methylated albumin. In either form, tritiated polypeptide was taken up by rabbit peritoneal exudate cells, composed largely of macrophages, in situ or in the test tube, and was found at equal levels in ribonucleic acid (RNA) purified by four extractions with phenol and repeated precipitation with ethanol. The label was found exclusively in the 4-5S RNA fraction, which was the only type perceptibly synthesized after the introduction of polypeptide, determined by incorporation of uridine-14C. There was no association between RNA and polypeptide mixed in vitro, but association comparable to that obtained with intact cells occurred with cell-free extracts. By using 35S-labeled T2 bacteriophage instead of polypeptide, antigen was again found only in 4-5S RNA. After total hydrolysis of RNA-polypeptide complexes followed by high-voltage electrophoresis on DEAE-cell-ulose paper, the 3H label was found only in glutamic acid. Using uridine-14C-labeled RNA, it was shown that the associated antigen was still in a polymer form by treatment of the complex with RNase followed by molecular sieving. The labeled polypeptide could not be dissociated from RNA by sucrose density gradient centrifugation, adsorption chromatography, gel electrophoresis, equilibrium sedimentation in cesium sulfate, or in competition by 104-105fold excesses of unlabeled poly-γ-D-glutamic acid, poly-α-D-glutamic acid, poly-α-L-glutamic acid, or D-glutamic acid. These results indicate that the association of antigen with macrophage RNA is an active biologic process and that the association is strong and possibly covalent. Association with macrophage RNA does not appear to distinguish between antigens on the basis of immunogenicity.
Fed-batch cultures of Bacillus licheniformis produced poly--glutamic acid (PGA), a water-soluble biodegradable polymer. PGA reached 35gl–1 with a productivity of 1gl–1h–1 by pulsed-feeding of citric acid (1.44gh–1) and l-glutamic acid (2.4gh–1) when citric acid was depleted from the culture medium.
Poly(-glutamic acid) (PGA) production in Bacillus subtilis IFO3335 was studied. When l-glutamic acid, citric acid, and ammonium sulfate were used as carbon and nitrogen sources, a large amount of PGA without a by-product such as a polysaccharide was produced. The time courses of cell growth, PGA, glutamic acid, and citric acid concentrations during cultivation were investigated. It was found that glutamic acid added to the medium was apparently not assimilated. It can be presumed that the glutamic acid unit in PGA is mainly produced from citric acid and ammonium sulfate. The PGA productivity was investigated at various concentrations of ammonium sulfate in the media, which caused the depression of cell growth, high productivity of PGA, and the production of PGA with a high relative molecular mass. The yield of PGA determined by gel permeation chromatography (GPC) reached approximately 20 g/l. This yield was the highest value for PGA production by B. subtilis IFO3335, suggesting that B. subtilis IFO3335 was a bacterium that could produce PGA effectively. Time courses relative to the molecular mass of PGA at various concentrations of ammonium sulfate were investigated. It was suggested that B. subtilis IFO3335 excreted a PGA degradation enzyme with the progress of cultivation and that PGA was degraded by this enzyme.
We found glutamate racemase activity in cell extracts of Bacillus subtilis IFO 3336, which abundantly produces poly-gamma-glutamate. The highest activity was obtained in the early stationary phase of growth. The racemase was purified to homogeneity. The enzyme was a monomer with a molecular mass of about 30 kDa and required no cofactor. It almost exclusively catalyzed the racemization of glutamate; other amino acids, including alanine and aspartate but not homocysteinesulfinate, were inactive as either substrates or inhibitors. Although the Vmax value of the enzyme for L-glutamate is 21-fold higher than that for D-glutamate, the Vmax/Km value for L-glutamate is almost equal to that for the D-enantiomer. The racemase gene, glr, was cloned into Escherichia coli cells and sequenced. The racemase was overproduced in the soluble fraction of the E. coli clone cells with the substitution of ATG for TTG, the initial codon of the glr gene. D-Amino acid aminotransferase activity was not detected in Bacillus subtilis IFO 3336 cells. B. subtilis CU741, a leuC7 derivative of B. subtilis 168, showed lower glutamate racemase activity and lower productivity of poly-gamma-glutamate than B. subtilis IFO 3336. These results suggest that the glutamate racemase is mainly concerned in D-glutamate synthesis for poly-gamma-glutamate production in B. subtilis IFO 3336.