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Munishwar N Gupta - One of the best experts on this subject based on the ideXlab platform.
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Refolding of urea denatured ovalbumin with three Phase Partitioning generates many conformational variants.
International journal of biological macromolecules, 2013Co-Authors: Gulam M. Rather, Munishwar N GuptaAbstract:Three Phase Partitioning is a process in which mixing t-butanol with ammonium sulphate with a protein solution leads to the formation of three Phases. Generally, the interfacial protein precipitate (formed between upper t-butanol rich and lower aqueous Phase) can be easily dissolved back in aqueous buffers. In case of ovalbumin, this led to a precipitate which was insoluble in aqueous buffers. This precipitate when solubilized with 8 M urea and subjected to three Phase Partitioning under various conditions led to many refolded soluble conformational variants of ovalbumin. One of these showed trypsin inhibitory activity, had marginally higher β-sheet content and had higher surface hydrophobicity (both with respect to native ovalbumin). Scanning electron microscopy and Atomic force microscopy of this preparation showed a thread like structure characteristic of amyloid fibrils. The behaviour of ovalbumin during three Phase Partitioning makes it a valuable system for gaining further understanding of protein aggregation.
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Three Phase Partitioning leads to subtle structural changes in proteins.
International journal of biological macromolecules, 2013Co-Authors: Gulam M. Rather, Munishwar N GuptaAbstract:Three Phase Partitioning consists of precipitation of proteins due to simultaneous presence of ammonium sulphate and t-butanol. The technique has been successfully used for purification and refolding of proteins. There are however indications that the structures of proteins subjected to three Phase Partitioning are different from native structure of proteins. Taking several proteins, the present work examines the structural changes in proteins by comparing their thermal stabilities, secondary structure contents, surface hydrophobicities, hydrodynamic radii and solubilities in the presence of ammonium sulphate. The results show that while the nature or extent of structural changes may vary, in all the cases the changes are rather subtle and not drastic in nature. Hence, the technique can be safely used for protein purification and refolding.
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a novel process for extraction of edible oils enzyme assisted three Phase Partitioning eatpp
Bioresource Technology, 2007Co-Authors: Ruchi Gaur, Aparna Sharma, S K Khare, Munishwar N GuptaAbstract:Abstract Three Phase Partitioning (TPP), a technique used in protein purification has been evaluated, for extraction of oil from three different plant sources viz: mango kernel, soybean and rice bran. The process consists of simultaneous addition of t -butanol (1:1, v/v) and ammonium sulphate (w/v) to a crude preparation/slurry. Under optimized condition, the protein appears as an interfacial precipitate between upper t -butanol containing oil and lower aqueous Phase. Pretreatment of the slurries with a commercial enzyme preparation of proteases, Protizyme™, followed by three Phase Partitioning resulted in 98%, 86% and 79% (w/w) oil yields in case of soybean, rice bran and mango kernel, respectively. The efficiency of the present technique is comparable to solvent extraction with an added advantage of being less time consuming and using t -butanol which is a safer solvent as compared to n -hexane used in conventional oil extraction process.
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Refolding of a denatured α-chymotrypsin and its smart bioconjugate by three-Phase Partitioning
Biocatalysis and Biotransformation, 2007Co-Authors: Meryam Sardar, Aparna Sharma, Munishwar N GuptaAbstract:α-Chymotrypsin inactivated with 8 M urea and 100 mM dithiothreitol could be completely reactivated by subjecting it to three-Phase Partitioning (TPP). TPP consisted of adding 30% w/v ammonium sulfate and t-butanol (volume equivalent to aqueous solution of denatured α-chymotrypsin) at 25°C. The activated α-chymotrypsin was recovered as an interfacial precipitate between the upper organic and lower aqueous Phase. It was found that this could be extended to a thermally inactivated smart bioconjugate of α-chymotrypsin with Eudragit S-100 (a reversibly soluble–insoluble methmethacrylate). The thermally inactivated bioconjugate had to be further subjected to urea and dithiothreitol before refolding by three-Phase Partitioning. Ninety per cent of the activity of the bioconjugate could be recovered. The free enzyme and its bioconjugate which lost activity in the presence of 90% dioxane recovered 94 and 90% of their activities, respectively, by employing TPP. The refolded free enzyme and its bioconjugate were eval...
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extraction of oil from jatropha curcas l seed kernels by enzyme assisted three Phase Partitioning
Industrial Crops and Products, 2004Co-Authors: Shweta Shah, Aparna Sharma, Munishwar N GuptaAbstract:Three Phase Partitioning has been evaluated for extraction of oil from Jatropha curcas L. seeds. This process consisted of simultaneous addition of t-butanol (1:1, v/v) and 30% (w/v) ammonium sulphate to the slurry prepared from Jatropha seed kernels. Combination of sonication and enzyme treatment with a commercial preparation of fungal proteases at pH 9, led to 97% oil yield within 2 h.
Aparna Sharma - One of the best experts on this subject based on the ideXlab platform.
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a novel process for extraction of edible oils enzyme assisted three Phase Partitioning eatpp
Bioresource Technology, 2007Co-Authors: Ruchi Gaur, Aparna Sharma, S K Khare, Munishwar N GuptaAbstract:Abstract Three Phase Partitioning (TPP), a technique used in protein purification has been evaluated, for extraction of oil from three different plant sources viz: mango kernel, soybean and rice bran. The process consists of simultaneous addition of t -butanol (1:1, v/v) and ammonium sulphate (w/v) to a crude preparation/slurry. Under optimized condition, the protein appears as an interfacial precipitate between upper t -butanol containing oil and lower aqueous Phase. Pretreatment of the slurries with a commercial enzyme preparation of proteases, Protizyme™, followed by three Phase Partitioning resulted in 98%, 86% and 79% (w/w) oil yields in case of soybean, rice bran and mango kernel, respectively. The efficiency of the present technique is comparable to solvent extraction with an added advantage of being less time consuming and using t -butanol which is a safer solvent as compared to n -hexane used in conventional oil extraction process.
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Refolding of a denatured α-chymotrypsin and its smart bioconjugate by three-Phase Partitioning
Biocatalysis and Biotransformation, 2007Co-Authors: Meryam Sardar, Aparna Sharma, Munishwar N GuptaAbstract:α-Chymotrypsin inactivated with 8 M urea and 100 mM dithiothreitol could be completely reactivated by subjecting it to three-Phase Partitioning (TPP). TPP consisted of adding 30% w/v ammonium sulfate and t-butanol (volume equivalent to aqueous solution of denatured α-chymotrypsin) at 25°C. The activated α-chymotrypsin was recovered as an interfacial precipitate between the upper organic and lower aqueous Phase. It was found that this could be extended to a thermally inactivated smart bioconjugate of α-chymotrypsin with Eudragit S-100 (a reversibly soluble–insoluble methmethacrylate). The thermally inactivated bioconjugate had to be further subjected to urea and dithiothreitol before refolding by three-Phase Partitioning. Ninety per cent of the activity of the bioconjugate could be recovered. The free enzyme and its bioconjugate which lost activity in the presence of 90% dioxane recovered 94 and 90% of their activities, respectively, by employing TPP. The refolded free enzyme and its bioconjugate were eval...
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extraction of oil from jatropha curcas l seed kernels by enzyme assisted three Phase Partitioning
Industrial Crops and Products, 2004Co-Authors: Shweta Shah, Aparna Sharma, Munishwar N GuptaAbstract:Three Phase Partitioning has been evaluated for extraction of oil from Jatropha curcas L. seeds. This process consisted of simultaneous addition of t-butanol (1:1, v/v) and 30% (w/v) ammonium sulphate to the slurry prepared from Jatropha seed kernels. Combination of sonication and enzyme treatment with a commercial preparation of fungal proteases at pH 9, led to 97% oil yield within 2 h.
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Three Phase Partitioning of starch and its structural consequences
Carbohydrate Polymers, 2004Co-Authors: Kalyani Mondal, Aparna Sharma, Munishwar N GuptaAbstract:Three Phase Partitioning (TPP) of starch and its derivatives was carried out by adding tert-butanol to the aqueous solution of the polysaccharides in presence of ammonium sulfate. Starch and its modified forms could be recovered in more than 90% yield in the form of an interfacial layer between aqueous and organic Phases. Quantitative FT-IR spectral analysis of TPP-treated potato starch revealed a clear change in the IR absorption band of the hydroxyl groups indicating a decrease in hydrogen bonding. TPP-treated starch showed lower biodegradability with wheat germ α-amylase.
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three Phase Partitioning for simultaneous renaturation and partial purification of aspergillus niger xylanase
Biochimica et Biophysica Acta, 2004Co-Authors: Ipsita Roy, Aparna Sharma, Munishwar N GuptaAbstract:Three-Phase Partitioning (TPP) is carried out by mixing ammonium sulfate and t-butanol to obtain organic Phase, interfacial precipitate and aqueous Phase. It is shown that TPP of an 8 M urea/100 mM dithiothreitol-denatured xylanase preparation resulted in simultaneous renaturation and purification. This integrated novel approach gave recovery of 93% enzyme activity with 21-fold purification. The implications of this in the context of recovering activity from inclusion bodies are discussed.
Saroat Rawdkuen - One of the best experts on this subject based on the ideXlab platform.
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three Phase Partitioning and proteins hydrolysis patterns of alkaline proteases derived from fish viscera
Separation and Purification Technology, 2014Co-Authors: Sunantha Ketnawa, Soottawat Benjakul, Oscar Martinezalvarez, Saroat RawdkuenAbstract:Abstract In this study, the recovery alkaline proteases from farmed giant catfish viscera were isolated by using three-Phase Partitioning (TPP). Factors affecting Partitioning efficiency such as salts, solvent types, pH, and incubation temperatures were studied. Furthermore, the application of extracted alkaline proteases on proteins hydrolysis was also determined. The system consisted of crude enzyme extract:t-butanol 1:0.5 (w/v), 50% sodium citrate, pH 8.0 with incubation temperature of 25 °C provided the highest enzyme recovery (220%). The smear protein bands with molecular weight of 20, 24, and 215 kDa of TPP fractions appeared on the protein stained gel. Two major clear zones (24 and 130 kDa) in the interPhase were observed on casein-substrate gel electrophoresis. Extracted alkaline proteases showed relatively high effective in protein hydrolysis. As a result, TPP provided high enzyme recovery and could be applied to other enzymes. The obtained alkaline proteases can be further applied in preparation of protein hydrolysates.
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three Phase Partitioning of protease from calotropis procera latex
Biochemical Engineering Journal, 2010Co-Authors: Saroat Rawdkuen, Phanuphong Chaiwut, Punyawatt Pintathong, Soottawat BenjakulAbstract:Abstract Three-Phase Partitioning (TPP) was used to partially purify protease from the latex of Calotropis procera ( C. procera ). To optimize the TPP for protease isolation a ratio of crude extract to t -butanol, percent saturation of (NH 4 ) 2 SO 4 , and the cycle of TPP was required. The highest proteolytic recovery (first cycle) of 182% with a purification of 0.95 folds was obtained at the interPhase of the system comprising the ratio of the crude extract to t -butanol of 1.0:0.5 with the presence of 50% (NH 4 ) 2 SO 4 . The second cycle of TPP was prepared by adding of (NH 4 ) 2 SO 4 up to 65% (w/v) to the bottom Phase obtained from 30% (NH 4 ) 2 SO 4 –1.0:0.5 system of the first TPP. A purification of 6.92-fold was achieved with about 132% activity recovery. SDS-PAGE and zymography profiles revealed the substantial isolation of protease from C. procera latex by the TPP. The molecular weight of major protease was found to be around 28 kDa. The present study shows high interesting outcomes and could be used as a primary purification process in comparison with existing literature's values.
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extraction and three Phase Partitioning behavior of proteases from papaya peels
Process Biochemistry, 2010Co-Authors: Phanuphong Chaiwut, Punyawatt Pintathong, Saroat RawdkuenAbstract:Abstract Dried papaya peels exhibited superior proteolytic activity to the fresh peels. An extraction with phosphate buffer pH 7.0 greatly maintained proteolytic activity when compared to water. SDS-PAGE verified that the extracted dried papaya peels held a wide range of proteins. To optimize the three-Phase Partitioning (TPP) for isolating the papaya peel proteases required a ratio of crude extract to t -butanol, the (NH 4 ) 2 SO 4 concentration and the TPP cycles. The ratio of the crude extract to t -butanol of 1.0:0.5 with the presence of 20% (NH 4 ) 2 SO 4 resulted in the highest proteolytic recovery at 253.5%, and 15.8-fold of purification in the bottom Phase. The TPP was then optimized by adding up to 55% (NH 4 ) 2 SO 4 to the bottom Phase of the first step. A purification of 10.1-fold with about 89.4% recovery was obtained. This study showed the TPP can be effectively employed for the extraction of proteases from papaya peels.
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Three-Phase Partitioning of trypsin inhibitor from legume seeds.
Process Biochemistry, 2009Co-Authors: Richa Kusuma Wati, Soottawat Benjakul, Theerapong Theppakorn, Saroat RawdkuenAbstract:Abstract Three-Phase Partitioning (TPP) was used to isolate trypsin inhibitors from navy bean (NB), red kidney bean (RK) and adzuki bean (AZ) from the Royal Project Foundation in Thailand. The method was to mix the crude extract with solid ammonium sulfate (30% saturation, w/v) and tert -butanol ( t -butanol) in order to obtain the three Phases. The trypsin inhibitor was purified to 5-, 14- and 7-fold with 315%, 441% and 228% recovery for NB, RK and AZ, respectively. The SDS-PAGE showed the major inhibitor band with the molecular weights (MWs) of 132, 118 and 13 kDa for NB, RK and AZ, respectively. The fractions from NB and AZ showed higher pH stability compared to that of the RK, and they had the optimum pH ranges of 7–9. The highest relative inhibitory activity of the fractions of NB and RK were found at 50 °C, and all fractions were quite stable at 90 °C for 60 min of incubation. Increasing the concentration of salt (up to 3%, w/v) did not significantly decrease the inhibitory activity of all fractions ( p > 0.05). The trypsin inhibitors from the three legumes were unable to inhibit the autolysis of Pacific whiting and arrowtooth flounder muscles.
Andrew J. Daugulis - One of the best experts on this subject based on the ideXlab platform.
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Bioproduction of benzaldehyde in a solid–liquid two-Phase Partitioning bioreactor using Pichia pastoris
Biotechnology Letters, 2010Co-Authors: Ashu N. Jain, Tanya R. Khan, Andrew J. DaugulisAbstract:The bioproduction of benzaldehyde from benzyl alcohol using Pichia pastoris was examined in a solid–liquid two-Phase Partitioning bioreactor (TPPB) to reduce substrate and product inhibition. Rational polymer selection identified Elvax 40W as an effective sequestering Phase, possessing partition coefficients for benzyl alcohol and benzaldehyde of 3.5 and 35.4, respectively. The use of Elvax 40W increased the overall mass of benzaldehyde produced by approx. 300% in a 5 l bioreactor, relative to a single Phase biotransformation. The two-Phase system had a molar yield of 0.99, indicating that only minor losses occurred. These results provide a promising starting point for solid–liquid TPPBs to enhance benzaldehyde production, and suggest that multiple, targeted polymers may provide relief for transformations characterized by multiple inhibitory substrates/product/by-products.
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Bioproduction of benzaldehyde in a solid-liquid two-Phase Partitioning bioreactor using Pichia pastoris.
Biotechnology letters, 2010Co-Authors: Ashu N. Jain, Tanya R. Khan, Andrew J. DaugulisAbstract:The bioproduction of benzaldehyde from benzyl alcohol using Pichia pastoris was examined in a solid–liquid two-Phase Partitioning bioreactor (TPPB) to reduce substrate and product inhibition. Rational polymer selection identified Elvax 40W as an effective sequestering Phase, possessing partition coefficients for benzyl alcohol and benzaldehyde of 3.5 and 35.4, respectively. The use of Elvax 40W increased the overall mass of benzaldehyde produced by approx. 300% in a 5 l bioreactor, relative to a single Phase biotransformation. The two-Phase system had a molar yield of 0.99, indicating that only minor losses occurred. These results provide a promising starting point for solid–liquid TPPBs to enhance benzaldehyde production, and suggest that multiple, targeted polymers may provide relief for transformations characterized by multiple inhibitory substrates/product/by-products.
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Quantifying maintenance requirements from the steady-state operation of a two-Phase Partitioning bioscrubber.
Biotechnology and bioengineering, 2005Co-Authors: David R. Nielsen, Andrew J. Daugulis, P. James MclellanAbstract:An innovative method for directly and explicitly quantifying the maintenance energy requirements of pure cultures growing on volatile organic compound (VOC) substrates in a two-Phase Partitioning bioscrubber is described. Direct evidence of maintenance energy requirements of Achromobacter xylosoxidans Y234 is provided both through observed reductions in the macroscopic biomass-to-substrate yield with decreasing specific growth rates, but more remarkably through achievement of steady-state operation. The data conclusively show that maintenance activities do occur in the two-Phase Partitioning bioscrubber and clearly illustrate the importance of this phenomenon to the operation of this process, and similar bioreactor systems. While benzene was selected as the principal, sole substrate of interest in this study, ethylbenzene degradation experiments were also subsequently performed to illustrate and confirm the general applicability of the proposed technique, as well as the potential capabilities of the two-Phase Partitioning bioscrubber for the continuous treatment of waste gases containing various VOCs. The proposed method has been shown to generate maintenance energy estimates that are consistent with those obtained while employing more widely recognized estimation strategies, further validating its capabilities. The proposed steady-state mode also offers key operational advantages in terms of decreased disposal requirements in two-Phase Partitioning bioscrubbers. (c) 2005 Wiley Periodicals, Inc.
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phosphonium ionic liquids for degradation of phenol in a two Phase Partitioning bioreactor
Applied Microbiology and Biotechnology, 2005Co-Authors: M D Baumann, Andrew J. Daugulis, Philip G JessopAbstract:Six ionic liquids (ILs), which are organic salts that are liquid at room temperature, were tested for their biocompatibility with three xenobiotic-degrading bacteria, Pseudomonas putida, Achromobacter xylosoxidans, and Sphingomonas aromaticivorans. Of the 18 pairings, seven were found to demonstrate biocompatibility, with one IL (trihexyl(tetradecyl)phosphonium bis(trifluoromethylsulfonyl) amide) being biocompatible with all three organisms. This IL was then used in a two-Phase Partitioning bioreactor (TPPB), consisting of 1 l aqueous Phase loaded with 1,580 mg phenol and 0.25 l IL, inoculated with the phenol degrader P. putida. This initially toxic aqueous level of phenol was substantially reduced by phenol Partitioning into the IL Phase, allowing the cells to utilize the reduced phenol concentration. The Partitioning of phenol from the IL to the aqueous Phase was driven by cellular demand and thermodynamic equilibrium. All of the phenol was consumed at a rate comparable to that of previously used organic-aqueous TPPB systems, demonstrating the first successful use of an IL with a cell-based system. A quantitative 31P NMR spectroscopic assay for estimating the log P values of ILs is under development.
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Steady state and dynamic performance of a two-Phase Partitioning bioscrubber for the treatment of benzene gas
2005Co-Authors: David R. Nielsen, Andrew J. DaugulisAbstract:Biological treatment of waste gases contaminated with toxic volatile organic compounds has classically been achieved using such popular designs such as biofilters and single-Phase bioscrubbers. While these configurations have found their own successes, the shortcomings of their operation have led to improved processing strategies through the design and operation of the two-Phase Partitioning bioscrubber. A two-Phase Partitioning bioscrubber is comprised of a cell-containing aqueous Phase, as well as an organic Phase to sequester high concentrations of toxic substrates. Suitable organic Phases must be carefully selected to ensure that they are biocompatible, non-bioavailable, immiscible in the aqueous Phase, and nonvolatile, while preserving sub-inhibitory conditions in the aqueous Phase through favorable equilibrium Partitioning. The rate of substrate Partitioning mass transfer between the two liquid Phases should be sufficiently high such that it is controlled by the demands of the cells. High levels of bioactivity can, therefore, be preserved when treating high loads of toxic substrates through the addition of an appropriate organic Phase, as sub-inhibitory aqueous concentrations are ensured while not limiting the cells by mass transfer. Recent design improvements of the two-Phase Partitioning bioscrubber have led to higher benzene removal rates, improved oxygen transfer, and increased process stability in the face of feed disturbances. Meanwhile, improved operational strategies have capitalized on the phenomenon of cellular maintenance energy, a critical microbiological parameter, resulting in streamlined steady state operation and reduced expenses. Experimental results demonstrate the remarkable performance of the two-Phase Partitioning bioscrubber, able to maintain high removal efficiencies and superior elimination capacities whether applied via a constant benzene feed, or through dynamic feed variations including benzene spikes and step changes. The robustness of this process is also demonstrated through rapid recovery after restart from a simulated shutdown. The sustainability of this process and its effortless operation is demonstrated experimentally through a long-term treatment.
Gulam M. Rather - One of the best experts on this subject based on the ideXlab platform.
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Refolding of urea denatured ovalbumin with three Phase Partitioning generates many conformational variants.
International journal of biological macromolecules, 2013Co-Authors: Gulam M. Rather, Munishwar N GuptaAbstract:Three Phase Partitioning is a process in which mixing t-butanol with ammonium sulphate with a protein solution leads to the formation of three Phases. Generally, the interfacial protein precipitate (formed between upper t-butanol rich and lower aqueous Phase) can be easily dissolved back in aqueous buffers. In case of ovalbumin, this led to a precipitate which was insoluble in aqueous buffers. This precipitate when solubilized with 8 M urea and subjected to three Phase Partitioning under various conditions led to many refolded soluble conformational variants of ovalbumin. One of these showed trypsin inhibitory activity, had marginally higher β-sheet content and had higher surface hydrophobicity (both with respect to native ovalbumin). Scanning electron microscopy and Atomic force microscopy of this preparation showed a thread like structure characteristic of amyloid fibrils. The behaviour of ovalbumin during three Phase Partitioning makes it a valuable system for gaining further understanding of protein aggregation.
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Three Phase Partitioning leads to subtle structural changes in proteins.
International journal of biological macromolecules, 2013Co-Authors: Gulam M. Rather, Munishwar N GuptaAbstract:Three Phase Partitioning consists of precipitation of proteins due to simultaneous presence of ammonium sulphate and t-butanol. The technique has been successfully used for purification and refolding of proteins. There are however indications that the structures of proteins subjected to three Phase Partitioning are different from native structure of proteins. Taking several proteins, the present work examines the structural changes in proteins by comparing their thermal stabilities, secondary structure contents, surface hydrophobicities, hydrodynamic radii and solubilities in the presence of ammonium sulphate. The results show that while the nature or extent of structural changes may vary, in all the cases the changes are rather subtle and not drastic in nature. Hence, the technique can be safely used for protein purification and refolding.
