Expression and purification of horseradish peroxidase in insect larvae

Loustau, María N.; Romero, Lucía Virginia; Levin, Gustavo Javier; Magri, María Laura; López, María Gabriela; Taboga, Oscar; Cascone, Osvaldo; Miranda, Magdalena

doi:10.1016/j.procbio.2007.10.011

Cited by 15 publications

(26 citation statements)

References 22 publications

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“…Several methods have been developed for the isolation and purification of peroxidase from different sources (10)(11)(12)(13)(14)(24)(25)(26)(27)(28). The present study extends and improves further the earlier work on purification of peroxidase ( Table 4).…”

Section: Discussionsupporting

confidence: 70%

Purification of Peroxidase from Horseradish (Armoracia rusticana) Roots

Lavery

MacInnis

MacDonald

et al. 2010

J. Agric. Food Chem.

106

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Peroxidase (EC 1.11.1.7) from horseradish ( Armoracia rusticana ) roots was purified using a simple, rapid, three-step procedure: ultrasonication, ammonium sulfate salt precipitation, and hydrophobic interaction chromatography on phenyl Sepharose CL-4B. The preparation gave an overall yield of 71%, 291-fold purification, and a high specific activity of 772 U mg(-1) protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified enzyme was homogeneous and had a molecular weight of approximately 40 kDa. The isolated enzyme had an isoelectric point of 8.8 and a Reinheitszahl value of 3.39 and was stable when stored in the presence of glycerol at -20 degrees C, with >95% retention of original enzyme activity for at least 6 months. Maximal activity of purified horseradish peroxidase (HRP) was obtained under different optimized conditions: substrate (guaiacol and H(2)O(2)) concentrations (0.5 and 0.3 mM, respectively), type of buffer (50 mM phosphate buffer), pH (7.0), time (1.0 min), and temperature of incubation (30 degrees C). In addition, the effect of HRP and H(2)O(2) in a neutral-buffered aqueous solution for the oxidation of phenol and 2-chlorophenol substrates was also studied. Different conditions including concentrations of phenol/2-chlorophenol, H(2)O(2), and enzyme, time, pH, and temperature were standardized for the maximal activity of HRP with these substrates; under these optimal conditions 89.6 and 91.4% oxidations of phenol and 2-chlorophenol were obtained, respectively. The data generated from this work could have direct implications in studies on the commercial production of this biotechnologically important enzyme and its stability in different media.

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Section: Discussionsupporting

confidence: 70%

Purification of Peroxidase from Horseradish (Armoracia rusticana) Roots

Lavery

MacInnis

MacDonald

et al. 2010

J. Agric. Food Chem.

106

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“…These enzymes have been purified and studied from many plant sources; for example, oil palm (Deepa and Arumughan 2002), sweet potato tubers (Leon et al 2002), melon (Rodriguez-Lopez et al 2000), cauliflower (Koksal et al 2008), rubber tree (Wititsuwannakul et al 1997), baculovirus (Levin et al 2005) and insect larvae (Loustau et al 2008). In rubber trees ( Hevea brasiliensis Willd.…”

Section: Introductionmentioning

confidence: 99%

Hevea brasiliensis cell suspension peroxidase: purification, characterization and application for dye decolorization

et al. 2013

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Peroxidases are oxidoreductase enzymes produced by most organisms. In this study, a peroxidase was purified from Hevea brasiliensis cell suspension by using anion exchange chromatography (DEAE-Sepharose), affinity chromatography (Con A-agarose) and preparative SDS-PAGE. The obtained enzyme appeared as a single band on SDS-PAGE with molecular mass of 70 kDa. Surprisingly, this purified peroxidase also had polyphenol oxidase activity. However, the biochemical characteristics were only studied in term of peroxidase because similar experiments in term of polyphenol oxidase have been reported in our pervious publication. The optimal pH of the purified peroxidase was 5.0 and its activity was retained at pH values between 5.0–10.0. The enzyme was heat stable over a wide range of temperatures (0–60°C), and less than 50% of its activity was lost at 70°C after incubation for 30 min. The enzyme was completely inhibited by β-mercaptoethanol and strongly inhibited by NaN3; in addition, its properties indicated that it was a heme containing glycoprotein. This peroxidase could decolorize many dyes; aniline blue, bromocresol purple, brilliant green, crystal violet, fuchsin, malachite green, methyl green, methyl violet and water blue. The stability against high temperature and extreme pH supported that the enzyme could be a potential peroxidase source for special industrial applications.

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“…The purified HRP-C molecular mass was around 44 kDa, similar to that of the HRP extracted from the roots of A. rusticana, thus indicating that there was no protein degradation or modification in the glycosylation degree. We have previously reported HRP-C purification from R. nu larval extract by immobilised metal ion affinity chromatography (IMAC) (Loustau et al 2008). Despite its appropriate performance, this affinity-based technology is expensive, mainly due to the matrix cost and the use of imidazole at the elution step.…”

Section: Resultsmentioning

confidence: 99%

“…These structural properties complicate its recombinant expression in prokaryote systems (Smith et al 1990). In our laboratory, we focus on the baculovirus-insect cell system as being inexpensive for the expression of recombinant HRP-C and have recently reported the expression of HRP-C as a fusion protein in Rachiplusia nu (Loustau et al 2008) and Spodoptera frugiperda larvae .…”

Section: Introductionmentioning

confidence: 99%

“…S. frugiperda larvae are highly susceptible to infection with BVs injected into the haemocoel but resistant to oral infection (Haas-Stapleton et al 2003. In contrast, R. nu presents both oral and intrahaemocoelical susceptibility (Ferrer et al 2007;Loustau et al 2008;López et al 2010). Gong et al (2006) achieved an important increase in the expression level of the cholera toxin subunit B-insulin fusion protein by adding the PPHS element to the protein coding sequence.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2011

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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 V(max) and K(m) values of the recombinant HRP-C were similar to those of the HRP from Armoracia rusticana roots.

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Expression and purification of horseradish peroxidase in insect larvae

Cited by 15 publications

References 22 publications

Purification of Peroxidase from Horseradish (Armoracia rusticana) Roots

Purification of Peroxidase from Horseradish (Armoracia rusticana) Roots

Hevea brasiliensis cell suspension peroxidase: purification, characterization and application for dye decolorization

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

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