Obtainment of a highly concentrated pancreatic serine proteases extract from bovine pancreas by precipitation with polyacrylate

Lombardi, Julia; Valetti, Nadia Woitovich; Picó, Guillermo; Boeris, Valeria

doi:10.1016/j.seppur.2013.05.047

Cited by 18 publications

(15 citation statements)

References 28 publications

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“…As noted in this paper and related patent applications, essentially similar results are found at temperatures from 2 to 20°C, PAA MW 5000–15000, blood stabilized with different anticoagulants, and different kosmotropic salts (citrates or phosphates) and at varied pH (+/−1 unit). In agreement with Sternberg and Hershberger (1974) and Lombardi, Woitovich Valetti, Picó, and Valeria Boeris (2013) PAA plasma protein precipitates can be stored frozen and that the thawed precipitates can be readily resuspended. It has also been shown that the PAA precipitates, can be solubilised in different aqueous solutions, for example, distilled water (1 vol:1 vol) or phosphate buffered saline, and interface well with established downstream chromatographic processes previously optimized for use with cold ethanol precipitated protein fractions.…”

Section: Resultssupporting

confidence: 81%

“…As noted in this paper and related patent applications, essentially similar results are found at temperatures from 2 to 20°C, PAA MW 5000-15000, blood stabilized with different anticoagulants, and different kosmotropic salts (citrates or phosphates) and at varied pH (+/−1 unit). In agreement with Sternberg and Hershberger (1974) and Lombardi, Woitovich Valetti, Picó, and Valeria Boeris (2013) PAA plasma protein precipitates can be stored frozen and that the thawed precipitates can be readily resuspended.…”

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confidence: 84%

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Polyacrylic acid based plasma fractionation for the production of albumin and IgG: Compatibility with existing commercial downstream processes

McCann

Alstine

Martínez

et al. 2020

Biotech & Bioengineering

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Commercial fractionation of human plasma into immunoglobulin‐ and albumin‐rich fractions is often initiated with sequential cold ethanol‐based precipitation methods, which have changed little over the past 70 years. The required low temperature (−4 to −8°C) and high concentrations of ethanol 8–40%) necessitate large‐scale fixed processing lines, and major capital investment and operating costs. The resulting fractions are then further purified by ethanol based precipitation or chromatographic procedures to obtain the purified final product. Aqueous polyacrylic acid (PAA) based precipitation, which readily interfaces with existing downstream processing, could offer advantages with respect to cost, safety, environmental impact, and flexibility. Sequential precipitation with 7%, 12%, and 20% (w/v) solutions of PAA 8000 in the presence of a kosmotropic salt (sodium citrate) gave fibrinogen‐, immunoglobulin‐, and albumin‐rich fractions with 80–90% yield and 64%, 55%, and 82% purity, respectively. Further purification of the IgG‐rich precipitate by caprylic acid precipitation and anion exchange chromatography, achieved a target purity of >99%. This was also achieved for the downstream processing of the albumin‐rich precipitate using a two‐step ion exchange chromatographic procedure. This work shows that PAA precipitation can be used in place of cold ethanol precipitation to generate crude IgG and albumin fractions which can be purified to final products of acceptable purity.

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Section: Resultssupporting

confidence: 81%

mentioning

confidence: 84%

Polyacrylic acid based plasma fractionation for the production of albumin and IgG: Compatibility with existing commercial downstream processes

McCann

Alstine

Martínez

et al. 2020

Biotech & Bioengineering

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“…The protease from B. circulans M34 was mostly inhibited by PMSF; however, 80-90% enzyme activities were retained in the presence of DTT and urea. In this regard, PMSF causes sulfonation of active region serine residues; therefore serine-type protease completely loses proteolytic activity (Li et al, 2009;Lombardi et al, 2013). This suggests that this protease is of serine type.…”

Section: Kinetic Parameters Of Purified Enzymementioning

confidence: 99%

Purification and characterization of organic solvent stable serine alkaline protease from newly isolated Bacillus circulans M34

Sarı

Loğoğlu

Öktemer

2015

Biomedical Chromatography

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A protease from newly isolated Bacillus circulans M34 was purified by Q‐Sepharose anion exchange chromatography and Sepharose–bacitracin affinity chromatography followed by (NH4)2SO4 precipitation. The molecular mass of the purified enzyme was determined using SDS–PAGE. The optimum pH and temperature for protease activity were 11 and 50°C, respectively. The effect of various metal ions on protease activity was investigated. Alkaline protease from Bacillus circulans M34 wase activated by Zn2+, Cu2+ and Co2+ up to 31%. The purified protease was found to be stable in the organic solvents, surfactants and oxidizing agent. The substrate specificity of purified protease was investigated towards different substrates. The protease was almost completely inhibited by the serine protease inhibitor phenylmethanesulfonyl fluoride. The kinetic parameters of the purified protease, maximum rate (Vmax) and Michaelis constant (Km), were determined using a Lineweaver–Burk plot. Copyright © 2015 John Wiley & Sons, Ltd.

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“…As is the case of polyacrylate, which was used to isolate amylase from a fungi culture, a purification factor around 2 and recoveries of 70% were found . Also, proteases from fresh pancreas homogenate was isolated with a purity of 5‐fold and a recovery of 33% under the best conditions tested . Woitovich et al using Eudragits ® L100 and S100 (a polyacrylate derivate) purified peroxidase from a fresh extract of Raphanus sativus L , recovery of biological activity was 50% of the initial activity in the homogenate with 1.5‐fold increase in its specific activity.…”

Section: Pe‐p As Tool For the Downstream Processing Of Proteins And Imentioning

confidence: 99%

Polyelectrolytes–protein complexes: a viable platform in the downstream processes of industrial enzymes at scaling up level

Valetti

Brassesco

Picó

2016

J of Chemical Tech & Biotech

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Complexation between proteins and polyelectrolytes through electrostatic interactions gives either soluble or non‐soluble complexes, leading to phase separation. The formation and the stability of these complexes are influenced by pH, ionic strength, ratio between protein and polyelectrolyte and charge density of protein and polyelectrolyte. This review presented the advantages on the protein precipitation by adding a polyelectrolyte solution to a natural enzyme source (microbiological, vegetal or animal homogenate), over the classical precipitation methods. An enzyme can be precipitated like non‐soluble complex, separated and then re‐dissolved, thus the method works, at the same time, like concentrative and pre‐purification steps together. © 2016 Society of Chemical Industry

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Obtainment of a highly concentrated pancreatic serine proteases extract from bovine pancreas by precipitation with polyacrylate

Cited by 18 publications

References 28 publications

Polyacrylic acid based plasma fractionation for the production of albumin and IgG: Compatibility with existing commercial downstream processes

Polyacrylic acid based plasma fractionation for the production of albumin and IgG: Compatibility with existing commercial downstream processes

Purification and characterization of organic solvent stable serine alkaline protease from newly isolated Bacillus circulans M34

Polyelectrolytes–protein complexes: a viable platform in the downstream processes of industrial enzymes at scaling up level

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