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Bioactive Levan-Type Exopolysaccharide Produced by Pantoea agglomerans ZMR7: Characterization and Optimization for Enhanced Production
1Department of Biology, College of Science (for Women), University of Baghdad, Baghdad, Iraq
2Anbar Education Directors, Al-Anbar, Iraq
3Department of Applied Chemistry, College of Applied Science, University of Fallujah, Iraq
4Biotechnology Research Center, University of Al-Nahrain, Baghdad, Iraq
5Department of Botany and Microbiology, Faculty of Science, Cairo University, Giza 12613, Egypt
6Bionanotechnology Program, Faculty of Nanotechnology for Postgraduate Studies, Cairo University, Sheikh Zayed Branch Campus, Sheikh Zayed City, Giza 12588, Egypt
J. Microbiol. Biotechnol. 2021; 31(5): 696-704
Published May 28, 2021 https://doi.org/10.4014/jmb.2101.01025
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Graphical Abstract
Introduction
Levan is a homo-exopolysaccharide, composed of fructose molecules with a glucose residue linked by β-(2-6) glycosidic bonds at the end, and this backbone makes levan a unique biopolymer [1]. This biopolymer is produced from sucrose as a sole substrate by many types of microorganisms through the activity of a specific enzyme known as levansucrase (E.C. 2.4.1.10), and released as exopolysaccharides (EPS). Various bacterial genera such as
Materials and Methods
Isolation, Screening and Identification of EPS-Producing Bacteria
The rhizospheric soil samples of maize plants (
Production and Isolation of EPS
Microbial EPS production was performed using production medium [per liter of distilled water (DW): sucrose, 200 g; yeast extract 2.5 g; MgSO4·7H2O, 1.0 g; K2HPO4, 5.5 g; (NH4)2SO4, 1.0 g; pH 7.2] [22]. Bacterial inocula were prepared in 250-ml Erlenmeyer conical flasks containing 50 ml of nutrient broth. One loopful of overnight bacterial culture was inoculated in nutrient broth and incubated in a shaking incubator (100 rpm) for 48 h at 35 ± 2°C. The production media were inoculated with 5% (v/v) of the inoculum (7 × 106 CFU/ml) and incubated at 35± 2°C for 48 h with gentle agitation (100 rpm). After the incubation period, the culture was centrifuged for 10 min at 10,000 g and the cell-free supernatant was mixed with 2 volumes of ice-cold absolute ethanol and chilled at 4°C for 48 h. The precipitated EPS was harvested by centrifugation for 15 min at 15,000 g at 4°C and the pellets were re-dissolved in DW and deproteinized seven times with 1/5 volume of Sevag reagent (chloroform/n-butanol, 5:1 v/v)[23]. Thereafter, the aqueous layer containing the deproteinized EPS was collected, mixed with 2 volumes of ice-cold absolute ethanol and chilled at 4°C for 48 h. The re-precipitated EPS was collected by centrifugation for 15 min at 15,000 g at 4°C and the collected pellet was washed three times with 1.5 volumes of absolute cold ethanol. Afterwards, the pellets were dissolved in DW and dialyzed through a membrane with a 10-kDa cutoff against DW several times for three days, freeze-dried and weighed [5]. The EPS content was estimated by the hydrolysis of EPS samples in 0.1 M HCl for 1 h at 100°C and the content of EPS in the hydrolyzed solution was analyzed as D-fructose by the dinitrosalicylic acid method [24]. The obtained quantity of D-fructose was then divided by a factor of 1.11 to calculate the amount of levan. The strain designated ZMR7, exhibiting maximum levan production, was selected for further investigations [25, 26].
Characterization of the EPS
Thin-layer chromatography (TLC) of the acid-hydrolyzed biopolymer was conducted to investigate the composition of the EPS produced by
The presence of fructose in the EPS produced by
Optimization of Levan Production
The optimization of cultural parameters was carried out by determining the effect of individual factors and incorporating these at the optimum level before optimizing the next parameter and the quantity of the produced levan was estimated. The effect of sucrose concentration on levan production was assessed at various concentrations (50, 100, 200, 300, 400, and 500 g/l). The production media were inoculated with
Biological Activities of Levan Produced by P. agglomerans ZMR7
whereas, B (absorbance of blank), S (absorbance of the sample).
Statistical Analysis
The statistical analysis was conducted by IBM SPSS Statistics software 22 for Windows (IBM, USA) and GraphPad Prism version 7 (GraphPad Software, Inc., USA). All data were expressed as mean ± SD and statistical analysis was performed by one-way analysis of variance (ANOVA) of Duncan’s test and
Results
Isolation, Screening and Identification of EPS-Producing Bacteria
A total of 128 bacterial colonies of different shapes were isolated from rhizospheres of maize plants. Of these isolates, 16 strains exhibited mucoid and slimy colonies on sucrose-rich media, which could be an indication for levan production. ESP-producing candidates were picked and further examined by quantitative estimation of the produced EPS. Based on the result of quantitative screening for EPS production, the most promising strain, designated ZMR7 and exhibiting the maximum production of the EPS (9.35 g/l), was selected for further investigations. Based on 16S rRNA gene sequence analysis, the most efficient strain was identified as
-
Fig. 1.
Neighbor-joining phylogenetic dendrogram based on 16S rDNA sequences shows the relationship between P. agglomerans ZMR7 and the related taxa.
Characterization of EPS
To determine the monosaccharide composition, the EPS produced by
The chemical structure of the EPS produced by
-
Table 1 . Comparison between FTIR values of levan produced by
P. agglomerans , standard, and other bacteria.Chemical Groups Standard levan from Z. mobilis (cm-1)B. phenoliresistens Levan (cm-1)P. bovis sp. Levan (cm-1)B. megaterium Levan (cm-1)P. agglomerans Levan (cm-1)O-H 3319.26 3394.1 3423 3354.37 3417.68 C-H 2935.48 2932.23 2935 2934.52 2935.66 C=O 1722.31 1647.88 1636 1647.93 1639.49 References [25] [4] [32] [33] This study
-
Fig. 2.
The FTIR spectrum of the levan produced by rhizospheric P. agglomerans ZMR7.
The 1H-NMR spectrum of
-
Table 2 . 13C-NMR chemical shift signal values of levan produced by
P. agglomerans ZMR7 and other bacteria.Carbon atom 13C-NMR chemical shift (ppm) Standard levan from Z. mobilis B. megaterium GJT321B. methylotrophicus SK 21.002P. agglomerans ZMR7C-1 60.761 59.86 61.20 60.58 C-2 104.641 104.14 104.66 104.38 C-3 77.683 76.25 77.51 75.83 C-4 75.754 75.15 76.10 76.74 C-5 80.783 80.23 80.77 80.35 C-6 63.957 63.31 63.94 63.15 Reference [25] [33] [23] This study
-
Fig. 3.
The 1H-NMR (A) and 13C-NMR spectra of the levan produced by P. agglomerans ZMR7.
Optimization of Levan Production
Unequivocally, results revealed the stimulating impact of increased sucrose concentration on levan production by
-
Fig. 4.
Impact of sucrose concentration (A), various nitrogen sources (B), incubation temperature (C), initial pH (D) and incubation period (E) on levan production and conversion of sucrose to levan by Error bars represent standard deviations (SD). Columns headed by the same letter are not significantly different according to Duncan’s multiple range test (P. agglomerans ZMR7.p < 0.05).
To further investigate the optimal conditions of levan production, different incubation temperatures were evaluated. The results clarified that the optimal incubation temperature for levan production by
Antiproliferative, Antileishmanial, and Antioxidant Activities of Levan Produced by P. agglomerans ZMR7
Levan exhibited antiproliferative activities toward both investigated cell lines (Fig. 5A). The results revealed dose-dependent anticancer activities of levan against RD cells where the cytotoxicity effect increased by increasing the levan concentration. Notwithstanding, the levan produced by
-
Fig. 5.
Biological activity of levan produced by P. agglomerans ZMR7: Antiproliferative effect against MDA and RD cell lines using MTT assay (A), antiparasitic effect against promastigotes ofL. tropica (B), antioxidant activity (DPPH free radical scavenging ability) of levan produced byP. agglomerans ZMR7 and ascorbic acid as a standard (C).
To determine the antioxidant activity of levan produced by
Discussion
The newly isolated strain
Production medium development is an essential strategy to get a higher yield using any microbial strain [40]. Several factors affecting the production of levan were examined, with sucrose among the factors that enhanced the productivity of
Interestingly, maximum levan production was detected at 35°C although the optimum temperature for growth of
The inhibitory activity of levan EPS produced by
Conclusion
A new levan-producing strain of
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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Related articles in JMB

Article
Research article
J. Microbiol. Biotechnol. 2021; 31(5): 696-704
Published online May 28, 2021 https://doi.org/10.4014/jmb.2101.01025
Copyright © The Korean Society for Microbiology and Biotechnology.
Bioactive Levan-Type Exopolysaccharide Produced by Pantoea agglomerans ZMR7: Characterization and Optimization for Enhanced Production
Safaa A. S. Al-Qaysi1*, Halah Al-Haideri1, Sana M. Al-Shimmary1, Jasim M. Abdulhameed2, Othman I. Alajrawy3, Mohammad M. Al-Halbosiy4, Tarek A. A. Moussa5*, and Mohamed G. Farahat5,6
1Department of Biology, College of Science (for Women), University of Baghdad, Baghdad, Iraq
2Anbar Education Directors, Al-Anbar, Iraq
3Department of Applied Chemistry, College of Applied Science, University of Fallujah, Iraq
4Biotechnology Research Center, University of Al-Nahrain, Baghdad, Iraq
5Department of Botany and Microbiology, Faculty of Science, Cairo University, Giza 12613, Egypt
6Bionanotechnology Program, Faculty of Nanotechnology for Postgraduate Studies, Cairo University, Sheikh Zayed Branch Campus, Sheikh Zayed City, Giza 12588, Egypt
Correspondence to:Safaa Ahmed Al-Qaysi, Safaaa_bio@csw.uobaghdad.edu.iq
Tarek A. A. Moussa, tarekmoussa@yahoo.com
Abstract
Levan is an industrially important, functional biopolymer with considerable applications in the food and pharmaceutical fields owing to its safety and biocompatibility. Here, levan-type exopolysaccharide produced by Pantoea agglomerans ZMR7 was purified by cold ethanol precipitation and characterized using TLC, FTIR, 1H, and 13C NMR spectroscopy. The maximum production of levan (28.4 g/l) was achieved when sucrose and ammonium chloride were used as carbon and nitrogen sources, respectively, at 35°C and an initial pH of 8.0. Some biomedical applications of levan like antitumor, antiparasitic, and antioxidant activities were investigated in vitro. The results revealed the ability of levan at different concentrations to decrease the viability of rhabdomyosarcoma and breast cancer cells compared with untreated cancer cells. Levan appeared also to have high antiparasitic activity against the promastigote of Leishmania tropica. Furthermore, levan had strong DPPH radical scavenging (antioxidant) activity. These findings suggest that levan produced by P. agglomerans ZMR7 can serve as a natural biopolymer candidate for the pharmaceutical and medical fields.
Keywords: Levan, exopolysaccharide, characterization, antioxidant, antiparasitic, antiproliferation
Introduction
Levan is a homo-exopolysaccharide, composed of fructose molecules with a glucose residue linked by β-(2-6) glycosidic bonds at the end, and this backbone makes levan a unique biopolymer [1]. This biopolymer is produced from sucrose as a sole substrate by many types of microorganisms through the activity of a specific enzyme known as levansucrase (E.C. 2.4.1.10), and released as exopolysaccharides (EPS). Various bacterial genera such as
Materials and Methods
Isolation, Screening and Identification of EPS-Producing Bacteria
The rhizospheric soil samples of maize plants (
Production and Isolation of EPS
Microbial EPS production was performed using production medium [per liter of distilled water (DW): sucrose, 200 g; yeast extract 2.5 g; MgSO4·7H2O, 1.0 g; K2HPO4, 5.5 g; (NH4)2SO4, 1.0 g; pH 7.2] [22]. Bacterial inocula were prepared in 250-ml Erlenmeyer conical flasks containing 50 ml of nutrient broth. One loopful of overnight bacterial culture was inoculated in nutrient broth and incubated in a shaking incubator (100 rpm) for 48 h at 35 ± 2°C. The production media were inoculated with 5% (v/v) of the inoculum (7 × 106 CFU/ml) and incubated at 35± 2°C for 48 h with gentle agitation (100 rpm). After the incubation period, the culture was centrifuged for 10 min at 10,000 g and the cell-free supernatant was mixed with 2 volumes of ice-cold absolute ethanol and chilled at 4°C for 48 h. The precipitated EPS was harvested by centrifugation for 15 min at 15,000 g at 4°C and the pellets were re-dissolved in DW and deproteinized seven times with 1/5 volume of Sevag reagent (chloroform/n-butanol, 5:1 v/v)[23]. Thereafter, the aqueous layer containing the deproteinized EPS was collected, mixed with 2 volumes of ice-cold absolute ethanol and chilled at 4°C for 48 h. The re-precipitated EPS was collected by centrifugation for 15 min at 15,000 g at 4°C and the collected pellet was washed three times with 1.5 volumes of absolute cold ethanol. Afterwards, the pellets were dissolved in DW and dialyzed through a membrane with a 10-kDa cutoff against DW several times for three days, freeze-dried and weighed [5]. The EPS content was estimated by the hydrolysis of EPS samples in 0.1 M HCl for 1 h at 100°C and the content of EPS in the hydrolyzed solution was analyzed as D-fructose by the dinitrosalicylic acid method [24]. The obtained quantity of D-fructose was then divided by a factor of 1.11 to calculate the amount of levan. The strain designated ZMR7, exhibiting maximum levan production, was selected for further investigations [25, 26].
Characterization of the EPS
Thin-layer chromatography (TLC) of the acid-hydrolyzed biopolymer was conducted to investigate the composition of the EPS produced by
The presence of fructose in the EPS produced by
Optimization of Levan Production
The optimization of cultural parameters was carried out by determining the effect of individual factors and incorporating these at the optimum level before optimizing the next parameter and the quantity of the produced levan was estimated. The effect of sucrose concentration on levan production was assessed at various concentrations (50, 100, 200, 300, 400, and 500 g/l). The production media were inoculated with
Biological Activities of Levan Produced by P. agglomerans ZMR7
whereas, B (absorbance of blank), S (absorbance of the sample).
Statistical Analysis
The statistical analysis was conducted by IBM SPSS Statistics software 22 for Windows (IBM, USA) and GraphPad Prism version 7 (GraphPad Software, Inc., USA). All data were expressed as mean ± SD and statistical analysis was performed by one-way analysis of variance (ANOVA) of Duncan’s test and
Results
Isolation, Screening and Identification of EPS-Producing Bacteria
A total of 128 bacterial colonies of different shapes were isolated from rhizospheres of maize plants. Of these isolates, 16 strains exhibited mucoid and slimy colonies on sucrose-rich media, which could be an indication for levan production. ESP-producing candidates were picked and further examined by quantitative estimation of the produced EPS. Based on the result of quantitative screening for EPS production, the most promising strain, designated ZMR7 and exhibiting the maximum production of the EPS (9.35 g/l), was selected for further investigations. Based on 16S rRNA gene sequence analysis, the most efficient strain was identified as
-
Figure 1.
Neighbor-joining phylogenetic dendrogram based on 16S rDNA sequences shows the relationship between P. agglomerans ZMR7 and the related taxa.
Characterization of EPS
To determine the monosaccharide composition, the EPS produced by
The chemical structure of the EPS produced by
-
Table 1 . Comparison between FTIR values of levan produced by
P. agglomerans , standard, and other bacteria..Chemical Groups Standard levan from Z. mobilis (cm-1)B. phenoliresistens Levan (cm-1)P. bovis sp. Levan (cm-1)B. megaterium Levan (cm-1)P. agglomerans Levan (cm-1)O-H 3319.26 3394.1 3423 3354.37 3417.68 C-H 2935.48 2932.23 2935 2934.52 2935.66 C=O 1722.31 1647.88 1636 1647.93 1639.49 References [25] [4] [32] [33] This study
-
Figure 2.
The FTIR spectrum of the levan produced by rhizospheric P. agglomerans ZMR7.
The 1H-NMR spectrum of
-
Table 2 . 13C-NMR chemical shift signal values of levan produced by
P. agglomerans ZMR7 and other bacteria..Carbon atom 13C-NMR chemical shift (ppm) Standard levan from Z. mobilis B. megaterium GJT321B. methylotrophicus SK 21.002P. agglomerans ZMR7C-1 60.761 59.86 61.20 60.58 C-2 104.641 104.14 104.66 104.38 C-3 77.683 76.25 77.51 75.83 C-4 75.754 75.15 76.10 76.74 C-5 80.783 80.23 80.77 80.35 C-6 63.957 63.31 63.94 63.15 Reference [25] [33] [23] This study
-
Figure 3.
The 1H-NMR (A) and 13C-NMR spectra of the levan produced by P. agglomerans ZMR7.
Optimization of Levan Production
Unequivocally, results revealed the stimulating impact of increased sucrose concentration on levan production by
-
Figure 4.
Impact of sucrose concentration (A), various nitrogen sources (B), incubation temperature (C), initial pH (D) and incubation period (E) on levan production and conversion of sucrose to levan by Error bars represent standard deviations (SD). Columns headed by the same letter are not significantly different according to Duncan’s multiple range test (P. agglomerans ZMR7.p < 0.05).
To further investigate the optimal conditions of levan production, different incubation temperatures were evaluated. The results clarified that the optimal incubation temperature for levan production by
Antiproliferative, Antileishmanial, and Antioxidant Activities of Levan Produced by P. agglomerans ZMR7
Levan exhibited antiproliferative activities toward both investigated cell lines (Fig. 5A). The results revealed dose-dependent anticancer activities of levan against RD cells where the cytotoxicity effect increased by increasing the levan concentration. Notwithstanding, the levan produced by
-
Figure 5.
Biological activity of levan produced by P. agglomerans ZMR7: Antiproliferative effect against MDA and RD cell lines using MTT assay (A), antiparasitic effect against promastigotes ofL. tropica (B), antioxidant activity (DPPH free radical scavenging ability) of levan produced byP. agglomerans ZMR7 and ascorbic acid as a standard (C).
To determine the antioxidant activity of levan produced by
Discussion
The newly isolated strain
Production medium development is an essential strategy to get a higher yield using any microbial strain [40]. Several factors affecting the production of levan were examined, with sucrose among the factors that enhanced the productivity of
Interestingly, maximum levan production was detected at 35°C although the optimum temperature for growth of
The inhibitory activity of levan EPS produced by
Conclusion
A new levan-producing strain of
Conflict of Interest
The authors have no financial conflicts of interest to declare.
Fig 1.

Fig 2.

Fig 3.

Fig 4.

Fig 5.

-
Table 1 . Comparison between FTIR values of levan produced by
P. agglomerans , standard, and other bacteria..Chemical Groups Standard levan from Z. mobilis (cm-1)B. phenoliresistens Levan (cm-1)P. bovis sp. Levan (cm-1)B. megaterium Levan (cm-1)P. agglomerans Levan (cm-1)O-H 3319.26 3394.1 3423 3354.37 3417.68 C-H 2935.48 2932.23 2935 2934.52 2935.66 C=O 1722.31 1647.88 1636 1647.93 1639.49 References [25] [4] [32] [33] This study
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Table 2 . 13C-NMR chemical shift signal values of levan produced by
P. agglomerans ZMR7 and other bacteria..Carbon atom 13C-NMR chemical shift (ppm) Standard levan from Z. mobilis B. megaterium GJT321B. methylotrophicus SK 21.002P. agglomerans ZMR7C-1 60.761 59.86 61.20 60.58 C-2 104.641 104.14 104.66 104.38 C-3 77.683 76.25 77.51 75.83 C-4 75.754 75.15 76.10 76.74 C-5 80.783 80.23 80.77 80.35 C-6 63.957 63.31 63.94 63.15 Reference [25] [33] [23] This study
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