Direct comparison between ion-exchange chromatography and aqueous two-phase processes for the partial purification of penicillin acylase produced by E. coli

Aguilar, Oscar; Albiter, Verónica; Serrano‐Carreón, Leobardo; Rito‐Palomares, Marco

doi:10.1016/j.jchromb.2006.03.016

Cited by 66 publications

(31 citation statements)

References 23 publications

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“…19 The system tie-line length (TLL), which represents the length of the line that connects the compositions of the top and bottom phases in a phase diagram for a defined system, was calculated as described previously. 24 Predetermined quantities of stock solutions of potassium phosphate and poly(ethylene glycol) (PEG; Sigma Chemicals, St Louis, MO, USA) of nominal molecular weights 600, 1000, 1450 and 3350 g mol −1 were mixed with 0.2 g of fractionated soybean extracts (7S or 11S; with a protein concentration of 32 and 9.0 mg cm −3 , respectively) or 0.2 g of purified β-glucuronidase solution with 30 059 U cm −3 (Sigma Chemicals) to give the desired PEG/salt composition (Table 1) with a final weight of 2.0 g (the amount of fractionated soybean extracts or the purified protein solution added to the ATPS represent 10% w/w of the total system). The amount of GUS in the stock solution (30 059 U cm −3 ) was defined to ensure enzyme activity detection in the ATPS.…”

Section: Aqueous Two-phase Experimentsmentioning

confidence: 99%

“…The potential economic benefits of substitution of costly unit operations, such as chromatography, by ATPS without commitment of the yield, have previously been addressed and the same strategies can be applied to plant-made products. 7,24 Recently, the potential applicability of ATPS for integrated extractive partitioning applied to the recovery of a model recombinant protein expressed in maize and tobacco has been demonstrated. 7,25 However, the potential application of ATPS to process soybean extracts as a route to the recovery of recombinant proteins has yet to be addressed.…”

Section: Introductionmentioning

confidence: 99%

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Processing of soybean (Glycine max) extracts in aqueous two‐phase systems as a first step for the potential recovery of recombinant proteins

Aguilar

Rito‐Palomares

2007

J of Chemical Tech & Biotech

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BACKGROUND: The potential use of plants as production systems to establish bioprocesses has been established over the past decade. However, the lack of efficient initial concentration and separation procedures affect the generic acceptance of plants as economically viable systems. In this context the use of aqueous two-phase systems (ATPS) can provide strategies to facilitate the adoption of plants as a base for bioprocesses. Among the crops, soybeans (Glycine max) represent an attractive alternative since potentially they can produce high levels of recombinant protein. In this paper the processing of fractionated soybean extracts using ATPS is evaluated as a first step to recover recombinant proteins expressed in plants, using β-glucuronidase (GUS; E.C. 3.2.1.31) as a model protein.

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Section: Aqueous Two-phase Experimentsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Processing of soybean (Glycine max) extracts in aqueous two‐phase systems as a first step for the potential recovery of recombinant proteins

Aguilar

Rito‐Palomares

2007

J of Chemical Tech & Biotech

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“…For example, very recently, ATPS composed of PEG and sodium citrate was successfully used for textile effluent dye removal [12]. It has been proven by many researchers that ATPS is an efficient and economical process when compared to other separation processes like precipitation, chromatography, and so forth [13][14][15].…”

Section: Introductionmentioning

confidence: 99%

Optimization of Aqueous Two-Phase Systems for the Recovery of Soluble Proteins from Tannery Wastewater Using Response Surface Methodology

Selvaraj

Murty

2013

Journal of Engineering

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Aqueous two-phase system (ATPS) composed of polyethylene glycol 6000 (PEG 6000) and sodium citrate (SC) has been proposed to recover the valuable soluble proteins from tannery wastewater. A sequential optimization strategy which included fractional factorial design (fFD) and central composite design (CCD) was employed to enhance the recovery. From this strategy, a secondorder polynomial model was obtained for the protein recovery and it was validated. The optimum recovery was found as 93.46% when pH, NaCl concentration, and temperature were kept at 7.5, 0.1 M, and 33 ∘ C, respectively, for a phase system composed of 20% (w/w) PEG 6000-15% (w/w) SC. Thus the proposed ATPS can serve as an alternative to the conventional precipitation method to recover the soluble proteins from tannery wastewater.

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“…One explanation for this observation is that high molecular weight of PEG has high hydrophobicity and thus increases the salting-out characters that drive the partition of recombinant bromelain to the upper phase [22]. Conversely, low molecular weight of polymer attracts the migration of contaminant proteins to the top phase and causes a decrease in remaining free volume available for the recombinant bromelain to occupy [23]. Thus, the K E , PF, and enzyme activity recovery values at the top phase were significantly reduced.…”

Section: Aqueous Two-phase Systemmentioning

confidence: 99%

“…In terms of reusability, the resin can be re-used up to 100 times only, with an additional cost of using sodium hydroxide, sodium chloride, nickel sulphate, and EDTA for stripping and regenerating the resin after each run. In the case of ATPS, the polymer and salt cannot be recycled as they were part of the final purified enzyme [23]. Such economic and operation analysis provides understanding for further research, and the ATPS process is proven to be a costeffective, time-saving, and a higher recovery method that may be scaled up for industrial purpose.…”

Section: Comparison Of Purification By Atps and Chromatographic Technmentioning

confidence: 99%

COMPARISON OF PURIFICATION METHODS TO PURIFY RECOMBINANT BROMELAIN FROM Escherichia coli BL21-A1

Arshad¹,

Amid²,

Yusof³

et al. 2017

MJAS

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Recombinant bromelain is a cysteine protease that can be exploited for its protease activity in food and pharmaceutical applications. The aim of this study was to compare different purification methods aqueous two-phase system (ATPS), ammonium sulphate precipitation, ion exchange, affinity, and gel filtration chromatography) for the recombinant bromelain purification from Escherichia coli BL21-A1. From the SDS-PAGE analysis, all methods produced band with molecular weight between 50 to 55 kDa. Among the methods used, the ATPS purification consisting of 13% (w/w) of PEG6000 and 11% (w/w) potassium phosphate at pH 7.0 was chosen as the best purification method. The method produced 16.39 ± 0.03 % of yield, purification fold of 5.35 ± 0.11, and specific activity of 3.47 ± 0.11 unit/mg of recombinant bromelain. This proposed study can be used as a platform for large-scale downstream processing of recombinant bromelain in industry.Keywords: recombinant bromelain, purification, chromatography, aqueous two-phase system Abstrak Rekombinan bromelain adalah sisteina protease yang boleh dieksploitasi dalam aplikasi makanan dan farmaseutikal. Tujuan kajian ini adalah untuk membandingkan kaedah penulenan yang berbeza dengan menggunakan sistem akueus dua fasa (ATPS), pemendakan amonium sulfat, pertukaran ion, afiniti dan gel penapisan kromatografi untuk penulenan rekombinan bromelain daripada Escherichia coli BL21-A1. Daripada analisis SDS-PAGE, semua kaedah menghasilkan jalur dengan berat molekul antara 50 hingga 55 kDa. Antara kaedah yang digunakan, sistem akueus dua fasa (ATPS) yang terdiri daripada 13% (w/w) PEG6000 dan 11% (w/w) kalium fosfat pada pH 7.0 telah dipilih sebagai kaedah penulenan yang terbaik. Kaedah ini mendapat 16.39 ± 0.03% hasil, kadar penulenan 5.35 ± 0.11, dan aktiviti rekombinan bromelain sebanyak 3.47 ± 0.11 unit/mg. Kajian ini dicadangkan boleh digunakan sebagai platform untuk pemprosesan hiliran rekombinan bromelain secara besar-besaran dalam industri.Kata kunci: rekombinan bromelain, penulenan, kromatografi, sistem akueus dua fasa IntroductionBromelain is a plant protease that is commercially obtained from stem, fruit, and waste of pineapple plant [1]. Bromelain is notable for being used in multitude industrial applications such as meat tenderization, food, beverages, and pharmaceuticals, hence increases its market demand [2]. However, there is a rising argument whether pineapple

show abstract

Direct comparison between ion-exchange chromatography and aqueous two-phase processes for the partial purification of penicillin acylase produced by E. coli

Cited by 66 publications

References 23 publications

Processing of soybean (Glycine max) extracts in aqueous two‐phase systems as a first step for the potential recovery of recombinant proteins

Processing of soybean (Glycine max) extracts in aqueous two‐phase systems as a first step for the potential recovery of recombinant proteins

Optimization of Aqueous Two-Phase Systems for the Recovery of Soluble Proteins from Tannery Wastewater Using Response Surface Methodology

COMPARISON OF PURIFICATION METHODS TO PURIFY RECOMBINANT BROMELAIN FROM Escherichia coli BL21-A1

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