Investigation Of Poly-γ-glutamic Acid Production From Six Different Bacillus Strains
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S428 Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 [P-M.9] Nucleic acid delivery with chitosan hydroxybenzotriazole Praneet Opanasopit 1,∗ , Sunee Techaarpornkul 1 , Theerasak Rojanarata 1 , Tanasait Ngawhirunpat 1 , Uracha Ruktanonchai 1,2 1 Faculty of Pharmacy, Silpakorn University, Nakornpathom, Thailand 2 National Nanotechnology Center, Thailand Science Park, Pathumthani, Thailand Keywords: Chitosan; siRNA delivery; DNA delivery; Hydroxyben- zotriazole Gene delivery is one of the critical steps for gene therapy. Nonvi- ral delivery systems have been developed. Among those, polymers are the major type of the non-viral vectors investigated in the past decade. Chitosan (CS) has been used as a vector for gene and siRNA delivery. In addition, CS is biocompatible, biodegradable and non- toxic; therefore, it has been proposed as a safer alternative to other non-viral vectors such as cationic lipids and cationic polymers. The objective of this study was to investigate the transfection efficiency of chitosan hydroxybenzotriazole (CS-HOBT) for in vitro nucleic acid delivery. The results revealed that CS-HOBT was able to con- dense with DNA/siRNA. Illustrated by agarose gel electrophoresis, complete complexes of CS-HOBT/DNA were formed at weight ratio of above 3 whereas those of CS-HOBT/siRNA were formed at weight ratio of above 4 (CSMW 20 and 45 KDa) and above 2 (CSMW 200 and 460 KDa). Gel electrophoresis results indicated that binding of CS- HOBT and DNA or siRNA depended on the MW and weight ratio. The particle sizes of CS-HOBT/nucleic acid complexes were in nano-size. The highest transfection efficiency of CS-HOBT/DNA complex was found at a weight ratio of 2 with the lowest CSMW of 20 kDa. The CS-HOBT mediated siRNA silencing of the endogenous EGFP gene occurred maximally with 60% efficiency. The CS-HOBT/siRNA com- plex with the lowest CSMW 20 kDa at a weight ratio of 80 showed the strongest inhibition of gene expression. For cytotoxicity studies, over 80% the average cell viabilities of the complexes were observed by MTT assay. This study suggests that CS-HOBT is straightforward to prepare, safe and exhibits significantly improved nucleic acid delivery potential in vitro. doi:10.1016/j.jbiotec.2010.09.597 [P-M.10] Investigation Of Poly--glutamic Acid Production From Six Dif- ferent Bacillus Strains A. Bhat 1,∗ , G. Kedia 1 , T. Bartlett 1 , D. Hill 1 , D. Charalampopoulos 2 , I. Radecka 1 1 University of Wolverhampton, United Kingdom 2 University of Reading, United Kingdom Keywords: PGA; Biopolymer; Biosynthesis; Nanoparticle Poly--glutamic acid (-PGA) is a naturally occurring biopoly- mer made up of repeating units of glutamic acid. Since -PGA is biodegradable, non toxic and non immunogenic, it has been used in numerous applications in the medical, food and cosmetic indus- tries, among others. Specific properties of -PGA make it useful for different applications. Following optimization, -PGA has the potential to be used in other novel applications because of which it is important to investigate more bacterial strains for its produc- tion. This study describes the production of -PGA with 6 different strains of bacteria – B. subtilis ATCC 23856, B. subtilis ATCC 23857, B. subtilis ATCC 23858, B. subtilis ATCC 23859, B. licheniformis NCIMB 1525 and B. licheniformis NCIMB 6816 in shake flasks. The effect of different growth media (GS and E) on production and proper- ties of -PGA was investigated. All bacteria produced -PGA with yields ranging from 4.1 g/l to 22.3 g/l. E Medium produced an amor- phous and acid form of -PGA whereas a crystalline Na salt of -PGA was obtained with GS Medium. B. licheniformis NCIMB 1525 and B. licheniformis NCIMB 6816 produced high molecular weight - PGA (850-871 kDa) with a polydispersity <1.5. This study not only explores the production of -PGA with bacterial strains that have not been used for its production before, but also throws light on the properties of -PGA thus obtained with different media. This information can be used to test novel applications of -PGA. doi:10.1016/j.jbiotec.2010.09.598 [P-M.11] Phytochemical Screening of Genista numidica and Evaluation of Antimicrobial activity of its Extracts A. Djahoudi, O. Toubal ∗ , S. Ati Department of Pharmacy, Algeria Keywords: Genista numidica; Phytochemical screening; Antimicro- bial activity; Disinfection; Herbal medicine Phytochemical screening of Genista numidica Spach ssp numidica (Spach), Batt.,(a northeast Algerian endemic plant) showed a wealth in polyphenol compounds: flavonoids (method of Lee et al, 1995) and saponins (method of Applebaum et al, 1969) and a lack of tannins, alkaloids, terpenes and sterols. The tests were conducted on aerial parts (stem, leaves and flowers). Cartography of the distribution area of the species and its ecological aspect were apprehended. The interest of this study is that this species remains until then unscreened. The results of Aromatogram by the incorporation method of Muller-Hinton on solid medium (method of Bauer and Kirby, 1966) show significant antimicrobial activity of the 03 extracts: infu- sion, saponins and flavonoids; the infusion is indeed much more active on coccus-Gram positive of Staphylococcus, such as saponins demonstrate activity on colonization Acinetobacter bacteria when the flavonoids act on both coccus-gram positive than on oxida- tive gram negative bacilli, like Pseudomonas. The raw extract has an important antifungal activity on Candida albicans. This could open opportunities for using this species in the treatment of urinary infections, and as a disinfecting additive on nosocomial area. It would be important to seek if antimicrobial activities are linked to the origin of the extract (flower, leaf), the nature of the solvent and the used strain; so that different parts of the plant, should be treated separately; than phytochemical analysis by the CPG method will perhaps show qualitative and quantitative dissimilarities between them. doi:10.1016/j.jbiotec.2010.09.599
Transcript of Investigation Of Poly-γ-glutamic Acid Production From Six Different Bacillus Strains
S428 Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
[P-M.9]
Nucleic acid delivery with chitosan hydroxybenzotriazole
Praneet Opanasopit 1,∗, Sunee Techaarpornkul 1, TheerasakRojanarata 1, Tanasait Ngawhirunpat 1, Uracha Ruktanonchai 1,2
1 Faculty of Pharmacy, Silpakorn University, Nakornpathom, Thailand2 National Nanotechnology Center, Thailand Science Park,Pathumthani, ThailandKeywords: Chitosan; siRNA delivery; DNA delivery; Hydroxyben-zotriazole
Gene delivery is one of the critical steps for gene therapy. Nonvi-ral delivery systems have been developed. Among those, polymersare the major type of the non-viral vectors investigated in the pastdecade. Chitosan (CS) has been used as a vector for gene and siRNAdelivery. In addition, CS is biocompatible, biodegradable and non-toxic; therefore, it has been proposed as a safer alternative to othernon-viral vectors such as cationic lipids and cationic polymers. Theobjective of this study was to investigate the transfection efficiencyof chitosan hydroxybenzotriazole (CS-HOBT) for in vitro nucleicacid delivery. The results revealed that CS-HOBT was able to con-dense with DNA/siRNA. Illustrated by agarose gel electrophoresis,complete complexes of CS-HOBT/DNA were formed at weight ratioof above 3 whereas those of CS-HOBT/siRNA were formed at weightratio of above 4 (CSMW 20 and 45 KDa) and above 2 (CSMW 200 and460 KDa). Gel electrophoresis results indicated that binding of CS-HOBT and DNA or siRNA depended on the MW and weight ratio. Theparticle sizes of CS-HOBT/nucleic acid complexes were in nano-size.The highest transfection efficiency of CS-HOBT/DNA complex wasfound at a weight ratio of 2 with the lowest CSMW of 20 kDa. TheCS-HOBT mediated siRNA silencing of the endogenous EGFP geneoccurred maximally with 60% efficiency. The CS-HOBT/siRNA com-plex with the lowest CSMW 20 kDa at a weight ratio of 80 showedthe strongest inhibition of gene expression. For cytotoxicity studies,over 80% the average cell viabilities of the complexes were observedby MTT assay. This study suggests that CS-HOBT is straightforwardto prepare, safe and exhibits significantly improved nucleic aciddelivery potential in vitro.
doi:10.1016/j.jbiotec.2010.09.597
[P-M.10]
Investigation Of Poly-�-glutamic Acid Production From Six Dif-ferent Bacillus Strains
A. Bhat 1,∗, G. Kedia 1, T. Bartlett 1, D. Hill 1, D. Charalampopoulos 2,I. Radecka 1
1 University of Wolverhampton, United Kingdom2 University of Reading, United KingdomKeywords: PGA; Biopolymer; Biosynthesis; Nanoparticle
Poly-�-glutamic acid (�-PGA) is a naturally occurring biopoly-mer made up of repeating units of glutamic acid. Since �-PGA isbiodegradable, non toxic and non immunogenic, it has been usedin numerous applications in the medical, food and cosmetic indus-tries, among others. Specific properties of �-PGA make it usefulfor different applications. Following optimization, �-PGA has thepotential to be used in other novel applications because of whichit is important to investigate more bacterial strains for its produc-tion. This study describes the production of �-PGA with 6 differentstrains of bacteria – B. subtilis ATCC 23856, B. subtilis ATCC 23857, B.subtilis ATCC 23858, B. subtilis ATCC 23859, B. licheniformis NCIMB1525 and B. licheniformis NCIMB 6816 in shake flasks. The effect
of different growth media (GS and E) on production and proper-ties of �-PGA was investigated. All bacteria produced �-PGA withyields ranging from 4.1 g/l to 22.3 g/l. E Medium produced an amor-phous and acid form of �-PGA whereas a crystalline Na salt of �-PGAwas obtained with GS Medium. B. licheniformis NCIMB 1525 andB. licheniformis NCIMB 6816 produced high molecular weight �-PGA (850-871 kDa) with a polydispersity <1.5. This study not onlyexplores the production of �-PGA with bacterial strains that havenot been used for its production before, but also throws light onthe properties of �-PGA thus obtained with different media. Thisinformation can be used to test novel applications of �-PGA.
doi:10.1016/j.jbiotec.2010.09.598
[P-M.11]
Phytochemical Screening of Genista numidica and Evaluation ofAntimicrobial activity of its Extracts
A. Djahoudi, O. Toubal ∗, S. Ati
Department of Pharmacy, AlgeriaKeywords: Genista numidica; Phytochemical screening; Antimicro-bial activity; Disinfection; Herbal medicine
Phytochemical screening of Genista numidica Spach sspnumidica (Spach), Batt.,(a northeast Algerian endemic plant)showed a wealth in polyphenol compounds: flavonoids (methodof Lee et al, 1995) and saponins (method of Applebaum et al, 1969)and a lack of tannins, alkaloids, terpenes and sterols. The tests wereconducted on aerial parts (stem, leaves and flowers). Cartographyof the distribution area of the species and its ecological aspect wereapprehended. The interest of this study is that this species remainsuntil then unscreened.
The results of Aromatogram by the incorporation method ofMuller-Hinton on solid medium (method of Bauer and Kirby, 1966)show significant antimicrobial activity of the 03 extracts: infu-sion, saponins and flavonoids; the infusion is indeed much moreactive on coccus-Gram positive of Staphylococcus, such as saponinsdemonstrate activity on colonization Acinetobacter bacteria whenthe flavonoids act on both coccus-gram positive than on oxida-tive gram negative bacilli, like Pseudomonas. The raw extract hasan important antifungal activity on Candida albicans.
This could open opportunities for using this species in thetreatment of urinary infections, and as a disinfecting additive onnosocomial area. It would be important to seek if antimicrobialactivities are linked to the origin of the extract (flower, leaf), thenature of the solvent and the used strain; so that different partsof the plant, should be treated separately; than phytochemicalanalysis by the CPG method will perhaps show qualitative andquantitative dissimilarities between them.
doi:10.1016/j.jbiotec.2010.09.599
