María V. Miranda

Partition behaviour and purification of a Mucor bacilliformis acid protease in aqueous two-phase systems

Abstract The partitioning of a Mucor bacilliformis acids protease, a potential substitute for bovine chymosin in cheese manufacture, was accomplished in various aqueous two-phase systems in order to investigate how changes in factors such as PEG (poly(ethylene glycol) molecular weight, pH and sodium chloride concentration, can modify the partition coefficient value. PEG/Reppal and PEG-phosphate systems were evaluated, in the presence of contaminating material from the solid substrate fermentation of the microorganism. When PEG-phosphate systems were analysed, it was found that K AP depended strongly on the PEG molecular weight (at pH 5·0, an increase in PEG molecular weight from 600 to 20 000 leads to a decrease in the K value from > 50 to 0·1). A dependence between K AP and system pH was also noticed, this effect being important at lower/intermediate PEG molecular weight. When PEG 1540 was used, a V-shaped distribution of K AP values was obtained, with a minimum at pH 5·0 (K = 1·40) and maxima at pH values of 3·0 (K > 40) and 5·8 (K = 14). Furthermore, the addition of NaCl led to an increase in K AP (for PEG 3350/phosphate at pH 5·0, K increased from 1·1 to > 35 when 1·0 mol kg −1 NaCl was added). Suitable conditions for enzyme purification were found in PEG 3350-phosphate systems at pH 3·0 and NaCl 1·0 mol kg −1 (K AP > 35, K CP = 0·10) and PEG-Reppal at pH 3·0, NaCl 1·5 mol kg −1 (K AP = 13, K CP = 0·32). In these systems, proteinaceous and particulate contaminating materials precipitated and adsorbed at the interphase, thus yielding a clear upper phase containing the purified enzyme. Furthermore, direct extraction of the fermented was performed using a PEG 20 000-Reppal-NaCl system (K AP = 14, K CP = 0·19, PF (purification factor) = 5·9). The enzyme can be recovered in the PEG 20 000-rich phase and back-extracted by adding salt (K AP = 0·25, K CP = 1·10, PF = 1·7). This method provides a simpler process for leaching and purification of an enzyme produced by solid-state fermentation.

Immobilization of soybean seed coat peroxidase on polyaniline: Synthesis optimization and catalytic properties

Soybean seed coat peroxidase (SBP) was immobilized on various polyaniline-based polymers (PANI), activated with glutaraldehyde. The most reduced polymer (PANIG2) showed the highest immobilization capacity (8.2 mg SBP g−1 PANIG2). The optimum pH for immobilization was 6.0 and the maximum retention was achieved after a 6-h reaction period. The efficiency of enzyme activity retention was 82%. When stored at 4°C, the immobilized enzyme retained 80% of its activity for 15 weeks as evidenced by tests performed at 2-week intervals. The immobilized SBP showed the same pH-activity profile as that of the free SBP for pyrogallol oxidation but the optimum temperature (55°C) was 10°C below that of the free enzyme. Kinetic analysis show that the Km was conserved while the specific Vmax dropped from 14.6 to 11.4 µmol min−1 µg−1, in agreement with the immobilization efficiency. Substrate specificity was practically the same for both enzymes. Immobilized SBP showed a greatly improved tolerance to different organic solvents; while free SBP lost around 90% of its activity at a 50% organic solvent concentration, immobilized SBP underwent only 30% inactivation at a concentration of 70% acetonitrile. Taking into account that immobilized HRP loses more than 40% of its activity at a 20% organic solvent concentration, immobilized SBP performed much better than its widely used counterpart HRP.

Study of variables involved in horseradish and soybean peroxidase purification by affinity chromatography on concanavalin A-Agarose

Abstract Variables involved in adsorption and elution of horseradish and soybean seed peroxidases from a concanavalin A-Agarose matrix were studied. The effect of pH, ion strength and Ca 2+ /Mg 2+ concentration on maximum capacity and dissociation constant was assessed through adsorption isotherms. The effect of flow rate and peroxidase concentration on dynamic capacity was assessed through breakthrough curves. For the elution step, NaCl and α- d -methylmannopyranoside concentrations were optimised. The best conditions for soybean peroxidase adsorption were pH 5.0 and 1 mM Ca 2+ /Mg 2+ in the absence of salt, at a flow rate up to 3.2 cm/min and 3 mg/ml peroxidase concentration. Elution required the addition of 0.48 M α- d -methylmannopyranoside and 0.97 M NaCl. Under these conditions, the dynamic capacity was 16.9 mg/ml matrix and purification yield, 84.3%. In the case of horseradish peroxidase, the best adsorption conditions were pH 7.0, 5 mM ions and 0.75 M NaCl, at a flow rate up to 1.5 cm/min and a 4 mg/ml peroxidase concentration. Dynamic capacity was 9.6 mg/ml matrix, and elution required 0.36 M α- d -methylmannopyranoside to yield 75% of enzyme. The dynamic-to-static capacity ratios were 0.71 and 0.62 for horseradish and soybean peroxidases, respectively.

Partition behaviour of horseradish peroxidase isoenzymes in aqueous two-phase poly(ethyleneglycol)/phosphate systems

Partition of purified horseradish peroxidase isoenzymes in aqueous two-phase systems was not affected by pH changes, but tie-line length and NaCl addition greatly increased the partition coefficient of all three isoenzymes, the former having more influence than the latter. In all systems, K were higher for acidic than those for neutral and basic isoenzymes, and K for basic were the lowest.

Bovine lactoferrin purification from whey using Yellow HE‐4R as the chromatographic affinity ligand

The worldwide production of whey increases by around 186 million tons each year and it is generally considered as a waste, even when several whey proteins have important economic relevance. For its valorization, inexpensive ligands and integrated chromatography methods need to be developed for specific and low-cost protein purification. Here, we describe a novel affinity process with the dye Yellow HE-4R immobilized on Sepharose for bovine lactoferrin purification. This approach based on a low-cost ligand showed an efficient performance for the recovery and purification of bovine lactoferrin directly from whey, with a yield of 71% and a purification factor of 61.

Rachiplusia nu larva as a biofactory to achieve high level expression of horseradish peroxidase

A process based on orally-infected Rachiplusia nu larvae as biological factories for expression and one-step purification of horseradish peroxidase isozyme C (HRP-C) is described. The process allows obtaining high levels of pure HRP-C by membrane chromatography purification. The introduction of the partial polyhedrin homology sequence element in the target gene increased HRP-C expression level by 2.8-fold whereas it increased 1.8-fold when the larvae were reared at 27°C instead of at 24°C, summing up a 4.6-fold overall increase in the expression level. Additionally, HRP-C purification by membrane chromatography at a high flow rate greatly increase D the productivity without affecting the resolution. The Vmax and Km values of the recombinant HRP-C were similar to those of the HRP from Armoracia rusticana roots.

Partition of horseradish peroxidase in aqueous two-phase systems containing polyvinylpyrrolidone

Growing peroxidase utilisation in different industries encourages the search for high benefit/cost ratio purification methods such as aqueous two-phase partition. In this way, the partitioning behaviour of peroxidase from Armoracia rusticanaroots in polyvinylpirrolidone/Reppal and polyvinylpirrolidone/salt aqueous two-phase systems was investigated. Based on these results, a two-step purification process was developed. In the first system (polyvinylpyrrolidone K30/Reppal PES 200, pH 7.0), cell debris and some contaminating proteins were shifted to the bottom phase while peroxidase concentrated in the top phase. After discarding the bottom phase, the second step involved addition of magnesium sulphate thus forming a second aqueous two-phase system. At this step, the enzyme was extracted into the salt-rich bottom phase. The overall yield was 75% and the purification factor 7.3.

Nanotoxicological Effects of SiO2 Nanoparticles on Spodoptera frugiperda Sf9 Cells

The application of silica nanoparticles (NPs) in the biomedical field experienced a great development. The driving forces for these and future developments are the possibility to design NPs with homogeneous size and structure amenable to specific grafting. Moreover, it is possible to tune the characteristics of the NPs to meet the requirements of each specific cell and desired application. Herein, we analyzed the effect of silica NPs of various sizes and surface charge on the viability of Spodoptera frugiperda cells (Sf9 cell line) with the aim of extending the knowledge of possible toxicity of the NPs in the environment and development of new tools for insect control. Moreover, these results will also contribute to develop more effective systems for gene vectors delivery and recombinant proteins expression. Bare silica NPs of 14 nm, 380 nm and 1430 nm as well as amine-modified silica NPs of 131 nm and 448 nm were obtained by the Stöber method. The NPs were characterized by DLS and zeta potential measurements. The cell viability was assessed by the MTT test. It was observed that the 14 nm NPs possess the highest toxic effect. Indeed, after 24 h, the viability of the cells exposed to the lower concentration of NPs (0.12 mg/ml) was about 40% of the value obtained for the control cells not exposed to NPs. Moreover, the exposure to other negative charged NPs also causes a lower activity when compared with the control. Alternatively, lower concentrations of positive charged NPs (i.e.: 0.12 or 0.6 mg/ml) demonstrated to stimulate the proliferation of the cells and higher concentrations (i.e.: 7.2 mg/ml) did not present significant differences with the control. In conclusion, we have demonstrated that the NPs possess an effect that is highly influenced by the size, charge and concentration. Although, silica NPs are being used in the biomedical field, these results contribute to further understanding the risk that could be associated to nanoparticles and how these can be modified in order to meet the requirements of each desired application.

Immobilisation of soybean seed coat peroxidase on its natural support for phenol removal from wastewater

Soybean seed coat peroxidase (SBP; EC 1.11.1.7) was immobilised on its natural support, soybean seed coats, anticipating its use in phenol removal. Periodate and glutaraldehyde chemistries were assayed. Periodate failed to immobilise any SBP, whereas glutaraldehyde was effective. The optimum concentration of glutaraldehyde was found to be 1%. Immobilisation shifted the optimum pH for phenol removal from 4.0 to 6.0. Treated seed coat retained its activity over a 4-week period, and reusability assays showed that treated seed coats could be reused once for phenol removal. Polyethylene glycol (PEG) increased the stability of phenol degradation activity. In addition, the phenolic polymer was adsorbed on to seed coats, thus making removal of the polymeric product easier.

Recombinant protein purification using complementary peptides as affinity tags.

Affinity tags have become highly popular tools for purifying recombinant proteins from crude extracts by affinity chromatography. Besides, short peptides are excellent ligands for affinity chromatography, as they are not likely to cause an immune response in case of leakage into the product, they are more stable than antibodies to elution and cleaning conditions and they usually have very acceptable selectivity. Hydropathically complementary peptides designed de novo show enough selectivity to be used successfully as peptide ligands for protein purification from crude extracts. Recognition specificity and selectivity in the interaction between the complementary peptide pair His-Leu-Leu-Phe-Pro-Ile-Ile-Ile-Ala-Ala-Ser-Leu and Lys-Asn-Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe have been demonstrated by other authors. In this work, we designed a recombinant protein purification method using a peptide affinity tag that binds to a peptide-binding partner immobilized on a chromatographic matrix. The enhanced green fluorescent protein expressed (EGFP) in Escherichia coli was used as the model. The peptide Gly-Gly-Gly-His-Leu-Leu-Phe-Pro-Ile-Ile-Ile-Ala-Ala-Ser-Leu was synthesized by solid phase using the Fmoc chemistry and immobilized in NHS-Sepharose (PC-Sepharose). Gly residues were added as a spacer arm at the N terminus. The EGFP was expressed either with the fusion tag Lys-Asn-Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe on the C terminus (EGFP-CPTag) or without any fusion tag. After cell disruption, the extract was directly applied to the PC-Sepharose column equilibrated with 20mM sodium phosphate buffer, pH 7.0. The adsorbed EGFP-CPTag was then eluted with 1M Tris. The yield was 98% and the purification factor 4.6. By contrast, EGFP without tag pass through without interacting with the PC-Sepharose column. The method designed can be applied for the purification of other recombinant proteins.