The harnessing of peptide-monolith constructs for single step plasmid DNA purification
- Han, Ying Chao, You, Greg, Pattenden, Leonard Keith, Forde, Gareth
- Authors: Han, Ying Chao , You, Greg , Pattenden, Leonard Keith , Forde, Gareth
- Date: 2010
- Type: Text , Journal article
- Relation: Process Biochemistry Vol. 45, no. 2 (2010), p. 203-209
- Full Text:
- Reviewed:
- Description: The availability of synthetic peptides has paved the way for their use in tailor-made interactions with biomolecules. In this study, a 16mer LacI-based peptide was used as an affinity ligand to examine the scale up feasibility for plasmid DNA purification. First, the peptide was designed and characterized for the affinity purification of lacO containing plasmid DNA, to be employed as a high affinity ligand for the potential capturing of plasmid DNA in a single unit operation. It was found there were no discernible interactions with a control plasmid that did not encode the lacO nucleotide sequence. The dissociation equilibrium constant of the binding between the 16mer peptide and target pUC19 was 5.0 ± 0.5 × 10-8M as assessed by surface plasmon resonance. This selectivity and moderated affinity indicate that the 16mer is suitable for the adsorption and chromatographic purification of plasmid DNA. The suitability of this peptide was then evaluated using a chromatography system with the 16mer peptide immobilized to a customized monolith to purify plasmid DNA, obtaining preferential purification of supercoiled pUC19. The results demonstrate the applicability of peptide-monolith supports to scale up the purification process for plasmid DNA using designed ligands via a biomimetic approach. © 2009 Elsevier Ltd. All rights reserved.
- Authors: Han, Ying Chao , You, Greg , Pattenden, Leonard Keith , Forde, Gareth
- Date: 2010
- Type: Text , Journal article
- Relation: Process Biochemistry Vol. 45, no. 2 (2010), p. 203-209
- Full Text:
- Reviewed:
- Description: The availability of synthetic peptides has paved the way for their use in tailor-made interactions with biomolecules. In this study, a 16mer LacI-based peptide was used as an affinity ligand to examine the scale up feasibility for plasmid DNA purification. First, the peptide was designed and characterized for the affinity purification of lacO containing plasmid DNA, to be employed as a high affinity ligand for the potential capturing of plasmid DNA in a single unit operation. It was found there were no discernible interactions with a control plasmid that did not encode the lacO nucleotide sequence. The dissociation equilibrium constant of the binding between the 16mer peptide and target pUC19 was 5.0 ± 0.5 × 10-8M as assessed by surface plasmon resonance. This selectivity and moderated affinity indicate that the 16mer is suitable for the adsorption and chromatographic purification of plasmid DNA. The suitability of this peptide was then evaluated using a chromatography system with the 16mer peptide immobilized to a customized monolith to purify plasmid DNA, obtaining preferential purification of supercoiled pUC19. The results demonstrate the applicability of peptide-monolith supports to scale up the purification process for plasmid DNA using designed ligands via a biomimetic approach. © 2009 Elsevier Ltd. All rights reserved.
- Chaffee, Alan, Lay, Galinda, Marshall, Marc, Jackson, William, Fei, Yi, Verheyen, Vincent, Cassidy, Peter, Scott, Steven
- Authors: Chaffee, Alan , Lay, Galinda , Marshall, Marc , Jackson, William , Fei, Yi , Verheyen, Vincent , Cassidy, Peter , Scott, Steven
- Date: 2010
- Type: Text , Journal article
- Relation: Fuel Vol. 89, no. 11 (2010), p. 3241-3249
- Full Text: false
- Reviewed:
- Description: The structure of a suite of high-volatile, bituminous Surat Basin, Queensland coals has been investigated by a combination of analytical probes. These included elemental analyses, pyrolysis-gas chromatography-mass spectroscopy and Fourier transform infrared spectroscopy, together with a study of their liquefaction products in both tetralin and solvent free-tin catalysed hydrogenations. Samples were gathered across a 300 m depth interval intersected by the sampling well. Most techniques revealed clear but subtle differences in structure as a function of depth. The oils produced by solvent free-tin catalysed hydrogenation, however, showed very distinct dependence with depth and the waxy content, as indicated by 1H-NMR, could be correlated with the coal seam gas content
Toxicity of opal Australian paper weak black liquors towards actinobacillus succinogenes 130Z
- Authors: Halder, Dibyadeep
- Date: 2022
- Type: Text , Thesis , Masters
- Full Text:
- Description: The objective of this study was to gain a better understanding of Opal Australian Paper’s (AP’s) waste streams as a potential substrate for biomanufacturing. Three low-value process streams were included in this study: Continuous Kraft Mill (CKM) weak black liquor (WBL), Neutral Sulphite Semi-Chemical (NSSC) WBL and WBL extracted from NSSC clean-pulp (CP). The primary focus of the study was to determine the chemical composition and extent of toxicity of these WBLs towards Actinobacillus succinogenes 130Z. This genetically engineered bacterium can produce a valuable platform chemical, succinic acid, using various carbohydrate sources. This project sought to increase knowledge of AP’s WBL and its suitability as a biomanufacturing substrate. Literature has been reviewed on various pre-treatment pathways for this waste stream, A. succinogenes 130Z and its effectiveness on producing succinic acid, and the analytical techniques that have been used to answer the project objectives. The effectiveness of pre-treating the NSSC and CP WBLs was measured using the following characteristics: (a) the colour content (specific absorbance) using UV-Vis spectroscopy, (b) indicative yields of several organic constituents through pyrolysis-gas chromatography-mass spectroscopy, (c) the atomic H/C and S/C ratios using an elemental analysis and (d) toxicity of the samples using a combination of semi-quantitative (plating trials) and quantitative (determination of minimum bactericidal concentration) techniques. The impact of an additional pre-treatment step of granular activated carbon for NSSC WBL was also researched here. InfraRed spectroscopy and toxicity analysis helped to conclude that CKM WBL is extremely toxic to Actinobacillus succinogenes 130Z and contained negligible amounts of residual sugar units. Characterising the NSSC and CP WBLs at the molecular level revealed the untreated WBLs are rich in lignin-derived methoxyphenols (up to 160 mg/kg) and contained low concentrations of elemental sulphur (up to approx. 1 mg/kg). Infra-red spectroscopy and gas chromatography indicated that the fermentable sugars in the WBLs were very low (< 1.2 mg/kg) even after pre-treatment. Both methoxyphenols and sulphur are known to be toxic to many bacteria and A. succinogenes 130Z could not tolerate more than 0.45 mg/ml (db, dry basis) and 0.78 mg/ml (db) of NSSC and CP WBLs respectively. Employing Amberlite® XAD7HP resins to pre-treat the NSSC and CP WBLs reduced their toxicity 2- to 5-fold, enabling the bacterium to survive in about 2.5 mg/ml (db) and 1 mg/ml (db) of NSSC and CP WBLs respectively. One possible reason for this is the significant reduction in lignin content afforded by this pre-treatment technique. These results conclude that AP’s WBLs are toxic and need to undergo pre-treatment prior to utilisation as a substrate for succinic acid production via fermentation.
- Description: Masters by Research
- Authors: Halder, Dibyadeep
- Date: 2022
- Type: Text , Thesis , Masters
- Full Text:
- Description: The objective of this study was to gain a better understanding of Opal Australian Paper’s (AP’s) waste streams as a potential substrate for biomanufacturing. Three low-value process streams were included in this study: Continuous Kraft Mill (CKM) weak black liquor (WBL), Neutral Sulphite Semi-Chemical (NSSC) WBL and WBL extracted from NSSC clean-pulp (CP). The primary focus of the study was to determine the chemical composition and extent of toxicity of these WBLs towards Actinobacillus succinogenes 130Z. This genetically engineered bacterium can produce a valuable platform chemical, succinic acid, using various carbohydrate sources. This project sought to increase knowledge of AP’s WBL and its suitability as a biomanufacturing substrate. Literature has been reviewed on various pre-treatment pathways for this waste stream, A. succinogenes 130Z and its effectiveness on producing succinic acid, and the analytical techniques that have been used to answer the project objectives. The effectiveness of pre-treating the NSSC and CP WBLs was measured using the following characteristics: (a) the colour content (specific absorbance) using UV-Vis spectroscopy, (b) indicative yields of several organic constituents through pyrolysis-gas chromatography-mass spectroscopy, (c) the atomic H/C and S/C ratios using an elemental analysis and (d) toxicity of the samples using a combination of semi-quantitative (plating trials) and quantitative (determination of minimum bactericidal concentration) techniques. The impact of an additional pre-treatment step of granular activated carbon for NSSC WBL was also researched here. InfraRed spectroscopy and toxicity analysis helped to conclude that CKM WBL is extremely toxic to Actinobacillus succinogenes 130Z and contained negligible amounts of residual sugar units. Characterising the NSSC and CP WBLs at the molecular level revealed the untreated WBLs are rich in lignin-derived methoxyphenols (up to 160 mg/kg) and contained low concentrations of elemental sulphur (up to approx. 1 mg/kg). Infra-red spectroscopy and gas chromatography indicated that the fermentable sugars in the WBLs were very low (< 1.2 mg/kg) even after pre-treatment. Both methoxyphenols and sulphur are known to be toxic to many bacteria and A. succinogenes 130Z could not tolerate more than 0.45 mg/ml (db, dry basis) and 0.78 mg/ml (db) of NSSC and CP WBLs respectively. Employing Amberlite® XAD7HP resins to pre-treat the NSSC and CP WBLs reduced their toxicity 2- to 5-fold, enabling the bacterium to survive in about 2.5 mg/ml (db) and 1 mg/ml (db) of NSSC and CP WBLs respectively. One possible reason for this is the significant reduction in lignin content afforded by this pre-treatment technique. These results conclude that AP’s WBLs are toxic and need to undergo pre-treatment prior to utilisation as a substrate for succinic acid production via fermentation.
- Description: Masters by Research
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