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.This revised version was published online in October 2005 with corrections to the Cover Date.     

<Emphasis Type="Italic">Rachiplusia nu</Emphasis> 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 V_max and K_m values of the recombinant HRP-C were similar to those of the HRP from Armoracia rusticana roots.     

Ethnobotany of Horseradish (<Emphasis Type="Italic">Armoracia rusticana</Emphasis>, Brassicaceae) and Its Wild Relatives (<Emphasis Type="Italic">Armoracia</Emphasis> spp.): Reproductive Biology and Local Uses in Their Native Ranges

Ethnobotany of Horseradish ( Armoracia rusticana , Brassicaceae) and Its Wild Relatives ( Armoracia spp.): Reproductive Biology and Local Uses in Their Native Ranges. Horseradish (Armoracia rusticana) has been cultivated for its root for over 2,000 years. Today, commercial production takes place primarily in North America and Europe where A. rusticana is propagated exclusively vegetatively. Like many vegetatively-propagated crops, cultivated A. rusticana plants are generally sterile. Armoracia rusticana is one of three species in the genus: A. macrocarpa is native to the Central Danube River Basin, A. rusticana is putatively native to Eastern Europe, and A. sisymbrioides is native to Siberia. Documenting the ways in which the three Armoracia species are used in their putative native ranges and the reproductive biology of Armoracia in these regions will advance understanding of the role of humans in the evolution of sterility in A. rusticana. In this broad-scale ethnobotanical analysis, we document 1) which Armoracia species are used in different geographic regions, 2) the reproductive biology of cultivated and natural Armoracia populations, and 3) the uses of Armoracia species in their native areas. Fieldwork was conducted in Austria, Bulgaria, Romania, and Russia. Ethnobotanical data were collected through written questionnaires. In all areas visited, A. rusticana is the primary Armoracia species; however, there is regional variation in the way the plant is used. Armoracia rusticana is propagated vegetatively and is not found in the wild. Armoracia macrocarpa and A. sisymbrioides reproduce by seed in nature. These data provide an understanding of contemporary uses and management strategies of Armoracia species in their native ranges, demonstrate the differential reproductive biology of a clonally-propagated crop relative to its wild progenitors, and emphasize the importance of ethnobotanical data collection for crops and their wild relatives from a broad geographic range.     

Extractive fermentation in cloud point system for lipase production by Serratia marcescens ECU1010

Extractive microbial fermentation for production of lipase by Serratia marcescens ECU1010 has been carried out in cloud point system. The cloud point system is composed of mixture nonionic surfactants with a ratio of Triton X-114 to Triton X-45 4:1 in aqueous solution. The lipase prefers to partition into the surfactant rich phase (coacervate phase) whereas the cells and other hydrophilic proteins retain in the dilute phase of cloud point system. Thus, a concentration factor 4.2-fold and a purification factor 1.3-fold of the lipase have been achieved in the extractive fermentation process. This is the first report about extractive fermentation of proteins in cloud point system.     

Culture of transformed horseradish roots as a source of fusicoccin-like ligands

Endogenous fusicoccin (FC) or related substances were sought in horseradish (Armoracia rusticana P.) roots. An actively growing root culture was derived from plants transformed with Agrobacterium rhizogenes. The presence of FC-like substances in ethanolic extracts from roots was established in a radioreceptor binding assay with plasmalemmal FC receptors and in radioimmune analysis with an antiserum specific for FC A. FC-like ligands were found in the tissue and medium of aseptically grown culture.Abbreviations FC fusicoccin - GC/MS gas chromatography/mass spectrometry - RIA radioimmunoassay - RRA radioreceptor analysis - BSA bovine serum albumin - Mes 4-morpholineethanesulfonic acid - HPLC high performance liquid chromatography     

Control of the activity of plant plasma membrane MgATPase by the viscosity of the aqueous phase

The activity of plant plasma membrane (PM) MgATPase (EC 3. 6. 1. 35) was studied in PM vesicles purified from spring wheat (Triticum aestivum L. cv. Drabant) roots, winter wheat (Triticum aestivum L. cv. Martonvásári-8) roots, and soybean (Glycine max L. cv. Williams) hypocotyls by aqueous polymer two-phase partitioning. MgATPase from spring wheat roots was assayed at 23°C (a) in the absence and presence of Triton X-100, (b) in the presence of either 1 mM or 3 mM MgATP^2?, and in the presence of increasing concentration of sucrose from 10 mM upto 1. 2 M. Activity of MgATPase in PM vesicles from winter wheat roots was measured at 21°C at sucrose concentrations of 15 mM–1. 1 M in the presence of 3 mM MgATP^2? and absence of Triton X-100 only. MgATPase activity from soybean hypocotyls was studied (a) in the absence and presence of Triton X-100, (b) both at 21 and 31°C, in the presence of 3 mM MgATP^2? and varying concentrations of sucrose between 10 mM and 1. 1 M. In all cases, independently of the assay conditions and the source of PM, the MgATPase activity decreased with increasing sucrose concentration. Latency of the MgATPase activity depended only slightly on the concentration of sucrose. The Q₁₀ value for the MgATPase activity from soybean hypocotyls (and thereby the Arrhenius activation energy of the enzymatic reaction) was independent of the sucrose concentration and of the presence of Triton X-100. At optimal assay conditions, the ATP-hydrolyzing activity of plant PM MgATPase (v) was inversely proportional to the m-th power of the viscosity of aqueous phase (η) as predicted by the modified Kramers'theory of enzymatic catalysis: v ∝ (1/η)^m, where m is an empirical parameter between 0 and 1. For the activity of MgATPase in the three species studied, m varied between 0. 5 and 1. 1 in good agreement with the theory. We thus conclude that (a) the activity of integral membrane enzyme-proteins may be controlled not only by the property(ies) of the membrane lipid phase but also by the viscosity of the aqueous phase in the vicinity of such enzymes, and (b) the determination of vesicle sidedness based on enzyme latency may need a minor revision. Our interpretation is in agreement with the molecular dynamics approach of enzymatic catalysis worked out for soluble enzymes.     

A novel two-step extraction method with detergent/polymer systems for primary recovery of the fusion protein endoglucanase I–hydrophobin I

Extraction systems for hydrophobically tagged proteins have been developed based on phase separation in aqueous solutions of non-ionic detergents and polymers. The systems have earlier only been applied for separation of membrane proteins. Here, we examine the partitioning and purification of the amphiphilic fusion protein endoglucanase I_core–hydrophobin I (EGI_core–HFBI) from culture filtrate originating from a Trichoderma reesei fermentation. The micelle extraction system was formed by mixing the non-ionic detergent Triton X-114 or Triton X-100 with the hydroxypropyl starch polymer, Reppal PES100. The detergent/polymer aqueous two-phase systems resulted in both better separation characteristics and increased robustness compared to cloud point extraction in a Triton X-114/water system. Separation and robustness were characterized for the parameters: temperature, protein and salt additions. In the Triton X-114/Reppal PES100 detergent/polymer system EGI_core–HFBI strongly partitioned into the micelle-rich phase with a partition coefficient (K) of 15 and was separated from hydrophilic proteins, which preferably partitioned to the polymer phase. After the primary recovery step, EGI_core–HFBI was quantitatively back-extracted (K_{EGIcore–HFBI}=150, yield=99%) into a water phase. In this second step, ethylene oxide–propylene oxide (EOPO) copolymers were added to the micelle-rich phase and temperature-induced phase separation at 55°C was performed. Total recovery of EGI_core–HFBI after the two separation steps was 90% with a volume reduction of six times. For thermolabile proteins, the back-extraction temperature could be decreased to room temperature by using a hydrophobically modified EOPO copolymer, with slightly lower yield. The addition of thermoseparating co-polymer is a novel approach to remove detergent and effectively releases the fusion protein EGI_core–HFBI into a water phase.     

A novel membrane-based process to isolate peroxidase from horseradish roots: optimization of operating parameters

The optimization of operating parameters for the isolation of peroxidase from horseradish (Armoracia rusticana) roots with ultrafiltration (UF) technology was systemically studied. The effects of UF operating conditions on the transmission of proteins were quantified using the parameter scanning UF. These conditions included solution pH, ionic strength, stirring speed and permeate flux. Under optimized conditions, the purity of horseradish peroxidase (HRP) obtained was greater than 84 % after a two-stage UF process and the recovery of HRP from the feedstock was close to 90 %. The resulting peroxidase product was then analysed by isoelectric focusing, SDS–PAGE and circular dichroism, to confirm its isoelectric point, molecular weight and molecular secondary structure. The effects of calcium ion on HRP specific activities were also experimentally determined.     

The Non-Ionic Detergent Brij 58 Conserves the Structure of the Tonoplast H+-ATPase of Mesembryanthemum crystallinum L. During Solubilization and Partial Purification

The effects of solubilization with Triton X-100 or Brij 58 on the polypeptide composition and the substrate affinity of the tonoplast H⁺-ATPase of plants of Mesembryanthemum crystallinum performing C₃ photosynthesis or crassulacean acid metabolism (CAM) have been compared. Although all known subunits of the tonoplast H⁺-ATPase were present in the fraction of solubilized proteins after treatment with Brij 58 or Triton X-100, with Triton X-100 the apparent K_M value for ATP hydrolysis was increased by a factor of 1.8 and 1.5 in preparations from C₃ and CAM plants, respectively, even at low concentrations in contrast to treatment with Brij 58. This is explained by structural changes of the tonoplast H⁺-ATPase due to the Triton X-100 treatment. After solubilization with Brij 58 the tonoplast H⁺-ATPase was partially purified by Superose-6 size-exclusion FPLC. When Brij 58 was present, addition of lipids to the chromatography buffer was not necessary to conserve enzyme activity in contrast to previously described purification methods using Triton X-100. The substrate affinity of the partial purified H⁺-ATPase was similar to the substrate affinity obtained for ATP-hydrolysis of native tonoplast vesicles, indicating that the enzyme structure during partial purification was conserved by using Brij 58. The results underline that the lipid environment of the tonoplast H⁺-ATPase is important for enzyme structure and function.     

温度诱导双水相体系分离芦荟多糖的分配行为研究

本文研究了芦荟多糖在温度诱导双水相体系中的分配行为,考察了Triton-114的浓度、温度、酸度、盐的浓度等因素对芦荟多糖分配行为的影响。结果表明,芦荟多糖趋于分配在水相,当Triton-114浓度为4%,pH=3,温度50℃时,芦荟多糖在水相中的回收率达到最大,多糖的含量也由原来的68.39%增加到75.63%。实验还表明,无机盐对芦荟多糖的分配行为具有很大的影响。     

Purification and Developmental Analysis of the Major Anionic Peroxidase from the Seed Coat of Glycine max

We show that the majority of peroxidase activity in soybean (Glycine max var Williams 82) seeds is localized to the seed coat. A single isozyme is responsible for this activity and has been purified to electrophoretic homogeneity by successive chromatography on DEAE Sepharose Fast Flow, concanavalin A-Sepharose, and Sephadex G-75. The peroxidase exhibits a pl of 4.1, an apparent molecular mass of 37 kilodaltons, and has properties characteristic of a glycoprotein. The enzyme begins to accumulate approximately 21 days after anthesis and continues to do so throughout the maturation of the seed coat where it can represent at least 5% of the soluble protein in dry seed coats. Due to its localization in the seed, we propose that this isozyme may play a role in the hardening of the seed coat.     

The seed coat-specific expression of a subtilisin-like gene, SCS1, from soybean

A seed coat-specific gene, SCS1 (Seed Coat Subtilisin 1), from soybean, Glycine max [L.] Merill, has been identified and studied. The gene belongs to a small family of genes with sequence similarity to the subtilisins, which are serine proteases. Northern blot analysis showed that SCS1 RNA accumulates to maximal levels in seed coats at 12 days post anthesis, preceding the final stages of seed coat differentiation. The SCS1 RNA was not found in other tissues including embryos, seed pods, flowers, stems, roots or leaves. In-situ hybridization studies confirmed the temporal pattern of expression observed by Northern blot analysis and further revealed a restricted pattern of RNA accumulation in thick-walled parenchyma cells of the seed coats. These cells are important in the apoplastic translocation of nutrients en route to the embryo from the vascular tissues. The tissue-specific subtilisin-like gene may be required for regulating the differentiation of the thick-walled parenchyma cells. Received: 10 January 2000 / Accepted: 22 February 2000     

Excretion of peroxidase from horseradish hairy root in combination with ion supplementation

Summary The effect of ion-supplemented medium on peroxidase excretion from horseradish (Armoracia rusticana) hairy roots was studied. Supplementation of mannitol instead of ions revealed that the excretion was stimulated not by osmotic pressure in the medium but by ionic properties. Extracellular peroxidase activity per dry cell was proportionally correlated with the ionic strength of the cations. CaCl₂ or MgCl₂ was found to be the most effective agent for excretion among other combinations. CaCl₂ supplementation at the beginning of the culture caused higher peroxidase production in the medium without a significant loss of final cell mass compared with CaCl₂ addition during the culture. Repeated batch culture with 50 mM CaCl₂ supplementation allowed a continuous retention of cell viability over 149 days and produced a great amount of extracellular peroxidase, 12-fold higher than that achieved in a 40-day-old batch culture with 50 mM CaCl₂ supplementation. Correspondence to: T. Kobayashi     

Latent nitrate reductase activity is associated with the plasma membrane of corn roots

Latent nitrate reductase activity (NRA) was detected in corn (Zea mays L., Golden Jubilee) root microsome fractions. Microsome-associated NRA was stimulated up to 20-fold by Triton X-100 (octylphenoxy polyethoxyethanol) whereas soluble NRA was only increased up to 1.2-fold. Microsome-associated NRA represented up to 19% of the total root NRA. Analysis of microsomal fractions by aqueous two-phase partitioning showed that the membrane-associated NRA was localized in the second upper phase (U₂). Analysis with marker enzymes indicated that the U₂ fraction was plasma membrane (PM). The PM-associated NRA was not removed by washing vesicles with up to 1.0 M NACl but was solubilized from the PM with 0.05% Triton X-100. In contrast, vanadate-sensitive ATPase activity was not solubilized from the PM by treatment with 0.1% Triton X-100. The results show that a protein capable of reducing nitrate is embedded in the hydrophobic region of the PM of corn roots.Abbreviations L₁ first lower phase - NR nitrate reductase - NRA nitrate-reductase activity - PM plasma membrane - T:p Triton X-100 (octylphenoxy polyethoxyethanol) to protein ratio - U₂ second upper phase     

A novel two-step extraction method with detergent/polymer systems for primary recovery of the fusion protein endoglucanase I-hydrophobin I

Extraction systems for hydrophobically tagged proteins have been developed based on phase separation in aqueous solutions of non-ionic detergents and polymers. The systems have earlier only been applied for separation of membrane proteins. Here, we examine the partitioning and purification of the amphiphilic fusion protein endoglucanase I(core)-hydrophobin I (EGI(core)-HFBI) from culture filtrate originating from a Trichoderma reesei fermentation. The micelle extraction system was formed by mixing the non-ionic detergent Triton X-114 or Triton X-100 with the hydroxypropyl starch polymer, Reppal PES100. The detergent/polymer aqueous two-phase systems resulted in both better separation characteristics and increased robustness compared to cloud point extraction in a Triton X-114/water system. Separation and robustness were characterized for the parameters: temperature, protein and salt additions. In the Triton X-114/Reppal PES100 detergent/polymer system EGI(core)-HFBI strongly partitioned into the micelle-rich phase with a partition coefficient (K) of 15 and was separated from hydrophilic proteins, which preferably partitioned to the polymer phase. After the primary recovery step, EGI(core)-HFBI was quantitatively back-extracted (K(EGIcore-HFBI)=150, yield=99%) into a water phase. In this second step, ethylene oxide-propylene oxide (EOPO) copolymers were added to the micelle-rich phase and temperature-induced phase separation at 55 degrees C was performed. Total recovery of EGI(core)-HFBI after the two separation steps was 90% with a volume reduction of six times. For thermolabile proteins, the back-extraction temperature could be decreased to room temperature by using a hydrophobically modified EOPO copolymer, with slightly lower yield. The addition of thermoseparating co-polymer is a novel approach to remove detergent and effectively releases the fusion protein EGI(core)-HFBI into a water phase.     

Overexpression of phytochrome A enhances the light-induced formation of adventitious shoots on horseradish hairy roots

A full-length cDNA of the gene for phytochrome A from Arabidopsis thaliana was fused with the 35S promoter of cauliflower mosaic virus (CaMV35S-PHYA) and introduced into horseradish (Armoracia rusticana Gaert., Mey. et Scherb.) hairy roots. The phytochrome level in hairy roots transformed with CaMV35SPHYA was about three times greater than that in normal hairy roots and the rate of light-induced formation of adventitious shoots was also higher in the hairy roots transformed with CaMV35SPHYA. These results indicate that the light-induced formation of adventitious shoots on horseradish hairy roots is closely related to the phytochrome level. Received: 11 August 1998 / Revision received: 21 October 1998 / Accepted: 20 November 1998     

Optimalization of the peroxidase production by tissue cultures of horseradish <Emphasis Type="Italic">in vitro</Emphasis>

Tissue cultures of Armoracia rusticana L., both transformed with Agrobacterium rhizogenes and nontransformed, were screened for peroxidase activity. Most of the derived and tested strains exhibited 20 times higher activity [from 99 to 723 U g⁻¹(d.m.)] than the root of the intact plant [(30 U g⁻¹ (d.m.)]. The highest peroxidase activity was found in tumour culture growing on the medium without growth regulators. The influence of the addition of sugars and heavy metal ions in the medium on peroxidase production was tested. Increase in peroxidase activity was observed after cultivation of horseradish culture with cadmium, cobalt, nickel or lead ions.This work is supported by Grant Agency of Czech Republic Project No. 526/04/0135.     

Investigation of extractive microbial transformation in nonionic surfactant micelle aqueous solution using response surface methodology

Extractive microbial transformation of L-phenylacetylcarbinol (L-PAC) in nonionic surfactant Triton X-100 micelle aqueous solution was investigated by response surface methodology. Based on the Box–Behnken design, a mathematical model was developed for the predication of mutual interactions between benzaldehyde, Triton X-100, and glucose on L-PAC production. It indicated that the negative or positive effect of nonionic surfactant strongly depended on the substrate concentration. The model predicted that the optimal concentration of benzaldehyde, Triton X-100, and glucose was 1.2 ml, 15 g, and 2.76 g per 100 ml, respectively. Under the optimal condition, the maximum L-PAC production was 27.6 mM, which was verified by a time course of extractive microbial transformation. A discrete fed-batch process for verification of cell activity was also presented.     

Purification of the insulin receptor from human placental membranes

Insulin receptors were purified from human placental microsomal membranes by solubilisation with Triton X-100 followed by Sepharose 6B chromatography, phosphate gradient elution from hydroxyapatite and affinity chromatography on concanavalin A-Sepharose. 2000-fold purification was achieved with 63% overall recovery. The purified receptor gave a single band on 3.75% polyacrylamide (0.1% Triton X-100) gel electrophoresis. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis there was a major band at 75,000 and a minor band at 80,000 daltons. The purified receptor rechromatographed on Sepharose 6B with an apparent molecular weight of 300,000.     

Wound-induced expression of horseradish peroxidase

Peroxidases have been implicated in the responses of plants to physiological stress and to pathogens. Wound-induced peroxidase of horseradish (Armoracia rusticana) was studied. Total peroxidase activity was increased by wounding in cell wall fractions extracted from roots, stems and leaves of horseradish. On the other hand, wounding decreased the peroxidase activity in the soluble fraction from roots. The enzyme activities of the basic isozymes were induced by wounding in horseradish leaves based on data obtained by fractionation of crude enzyme in isoelectric focusing gel electrophoresis followed by activity staining. We have previously isolated genomic clones for four peroxidase genes, namely, prxC1a, prxC1b, prxC2 and prxC3. Northern blot analysis using gene-specific probes showed that mRNA of prxC2, which encodes a basic isozyme, accumulated by wounding, while the mRNAs for other peroxidase genes were not induced. Tobacco (Nicotiana tabacum) plants were transformed with four chimeric gene constructs, each consisting of a promoter from one of the peroxidase genes and the -glucuronidase (GUS) structural gene. High level GUS activity induced in response to wounding was observed in tobacco plants containing the prxC2-GUS construct.Abbreviations HRP horseradish peroxidase - prx gene for peroxidase - GUS -glucuronidase - CaMV cauliflower mosaic virus