Immobilisation of bromoperoxidase from Corallina officinalis

Summary The vanadium-dependent bromoperoxidase from the macroalga Corallina officinalis was immobilised on a cellulose acetate support with retention of approaching 50% of the applied units of activity. The enzyme exhibited high thermal stability and retained activity in repeated use. The immobilised enzyme showed tolerance to organic solvents similar to that of the free enzyme in the case of methanol but differed for acetone and ethanol, and with the latter showed enhanced activity as the % by volume of the solvent was increased.     

Efficient purification with high recovery of vanadium bromoperoxidase from Corallina officinalis

A novel, simple and highly efficient process for purifying vanadium bromoperoxidase from Corallina officinalis is reported. The key innovation is adding 0.5 mM sodium orthovanadate to the crude cell extract followed by heating at 70°C for 2 h, by which a 5.4-fold purification with a 100% activity recovery was achieved. Combining the heat treatment with ammonium sulfate precipitation and DEAE-52 column chromatography, the overall yield was 84%, 3.8 times greater than the highest yield previously reported. Finally, a specific activity of 310 U/mg, a 27-fold purification of the crude enzyme solution was produced.     

Crystal structure of dodecameric vanadium-dependent bromoperoxidase from the red algae Corallina officinalis

The three-dimensional structure of the vanadium bromoperoxidase protein from the marine red macroalgae Corallina officinalis has been determined by single isomorphous replacement at 2.3 A resolution. The enzyme subunit is made up of 595 amino acid residues folded into a single alpha+beta domain. There are 12 bromoperoxidase subunits, arranged with 23-point group symmetry. A cavity is formed by the N terminus of each subunit in the centre of the dodecamer. The subunit fold and dimer organisation of the Cor. officinalis vanadium bromoperoxidase are similar to those of the dimeric enzyme from the brown algae Ascophyllum nodosum, with which it shares 33 % sequence identity. The different oligomeric state of the two algal enzymes seems to reflect separate mechanisms of adaptation to harsh environmental conditions and/or to chemically active substrates and products. The residues involved in the vanadate binding are conserved between the two algal bromoperoxidases and the vanadium chloroperoxidase from the fungus Curvularia inaequalis. However, most of the other residues forming the active-site cavity are different in the three enzymes, which reflects differences in the substrate specificity and stereoselectivity of the reaction. A dimer of the Cor. officinalis enzyme partially superimposes with the two-domain monomer of the fungal enzyme.     

Purification of bromoperoxidase from Corallina pilulifera

Bromoperoxidase was purified from the crude extract of Corallina pilulifera (Corallinaeae, Rhodophyta) and found to be homogeneous upon disc gel electrophoresis by precipitation of ammonium sulfate and sequential column chromatographies of DEAE-Sepharose CL-6B, Sepharose 6B and Cellulofine GC-700m. The purified enzyme did not exhibit optical absorption spectra of a hemoprotein. Therefore, bromoperoxidase of C. pilulifera was completely distinguishable from other haloperoxidases which have heme-irons at the catalytic sites.     

Reactivity of recombinant and mutant vanadium bromoperoxidase from the red alga Corallina officinalis

Vanadium bromoperoxidase (VBPO) from the marine red alga Corallina officinalis has been cloned and heterologously expressed in Esherichia coli. The sequence for the full-length cDNA of VBPO from C. officinalis is reported. Steady state kinetic analyses of monochlorodimedone bromination reveals the recombinant enzyme behaves similarly to native VBPO from the alga. The kinetic parameters (K(m)(Br-)=1.2 mM, K(m)(H(2)O(2))=17.0 microM) at the optimal pH 6.5 for recombinant VBPO are similar to reported values for enzyme purified from the alga. The first site-directed mutagenesis experiment on VBPO is reported. Mutation of a conserved active site histidine residue to alanine (H480A) results in the loss of the ability to efficiently oxidize bromide, but retains the ability to oxidize iodide. Kinetic parameters (K(m)(I-)=33 mM, K(m)(H(2)O(2))=200 microM) for iodoperoxidase activity were determined for mutant H480A. The presence of conserved consensus sequences for the active sites of VBPO from marine sources shows its usefulness in obtaining recombinant forms of VBPO. Furthermore, mutagenesis of the conserved extra-histidine residue shows the importance of this residue in the oxidation of halides by hydrogen peroxide.     

Characterization of nonheme type bromoperoxidase in Corallina pilulifera

Bromoperoxidase was purified from the crude extract of Corallina pilulifera to be homogeneous upon polyacrylamide disc gel and sodium dodecyl sulfate-polyacrylamide gel electrophoreses according to the procedures previously reported (Itoh, N., Izumi, Y., and Yamada, H. (1985) Biochem. Biophys. Res. Commun. 131, 428-435). The enzyme had a molecular weight of approximately 790,000 and was composed of 12 subunits of identical molecular weights (Mr 64,000). Hexagonal molecular shapes of the enzyme were observed by electron microscopy. The isoelectric point of the enzyme was 3.0, and the predominance of acidic amino acids was revealed by amino acid analysis of the enzyme. The enzyme was specific for I- and Br- and inactive toward Cl- and F-. The optimum pH of the enzyme was 6.0, and the enzyme was stable in a range from pH 5.0 to 11.0. The enzyme had no hemeor flavin-like compounds as a prosthetic group. Plasma emission spectroscopy revealed that the enzyme contains 2.3 +/- 0.2 atoms of iron and 1.6 +/- 0.1 atoms of magnesium/molecule of protein. Hence, bromoperoxidase of C. pilulifera was distinct from other haloperoxidases and many peroxidases, which are hemoproteins.     

Characterization of nonheme iron and reaction mechanism of bromoperoxidase in Corallina pilulifera

The properties of the nonheme iron of bromoperoxidase from Corallina pilulifera were studied. The enzyme lost its activity when reduced with formamidine-sulfinic acid and recovered it when oxidized by air. Incubation of the enzyme with ferric or ferrous ion-chelating agents indicated that its nonheme iron was ferric. Analyses of circular dichroism and proton NMR spectra suggested that the ferric ion tightly bound to cysteine, histidine, or tyrosine residues of the enzyme. The enzyme catalyzed Br--dependent catalase reactions to yield 1 mol of O2 from 2 mol of H2O2. No O2 evolution was observed when bromination reaction of monochlorodimedone occurred. From these results, together with previous knowledge of this enzyme, it was concluded that it activated bromide anion (Br-) to bromonium cation (Br+) using one molecule of H2O2, and this Br+OH- formed at the active site then decomposed another H2O2 to yield O2 in the absence of halogen acceptors (substrate). When substrate was present in the reaction mixture, it and H2O2 competitively reacted with the reaction intermediate (Br+OH-) to give brominated products.     

Enhancing effect of calcium and vanadium ions on thermal stability of bromoperoxidase from Corallina pilulifera

Bromoperoxidase from the macro-alga Corallina pilulifera is an enzyme that possesses vanadate in the catalytic center, and shows a significant thermostability and stability toward organic solvents. The structural analysis of the recombinant enzyme overexpressed in yeast revealed that it contains one calcium atom per subunit. This has been confirmed by inductively coupled plasma emission spectrometry experiments. The study of the effect of metal ions on the apo-enzyme stability has shown that the calcium ion significantly increased the enzyme stability. In addition, vanadate also increased the thermostability and strontium and magnesium ions had similar effects as calcium. The holo-enzyme shows high stability in a range of organic solvents. The effect of the different ions and solvents on the structure of the enzyme has been studied by circular dichroism experiments. The high stability of the enzyme in the presence of organic solvents is useful for its application as a biocatalyst.     

In vitro antioxidant activities of sulfated polysaccharide fractions extracted from Corallina officinalis

Sulfated polysaccharides (F1, F2) from seaweed Corallina officinalis were isolated through anion-exchange column chromatography. Their chemical characteristics were determined by GC, HPLC, FT-IR and UV spectra. F1 and F2 contained only two monosaccharides, namely galactose and xylose. The antioxidant activities of F1, F2 and the de-sulfated polysaccharides (DF-1, DF-2) in vitro were investigated, including hydroxyl radicals scavenging effect, superoxide radical scavenging capacity, DPPH radical activity and reducing power. As expected, antioxidant assay showed that the two sulfated polysaccharide fractions (F1, F2) possessed considerable antioxidant properties and had more excellent abilities than de-sulfated polysaccharides (DF-1, DF-2).     

Substrate specificity, regiospecificity and stereospecificity of halogenation reactions catalyzed by non-heme-type bromoperoxidase of Corallina pilulifera

Many organic compounds were found to be substrates for halogenation reactions catalyzed by the non-heme-type bromoperoxidase found in the red alga Corallina pilulifera. Anisole, 1-methoxynaphthalene and thiophene were converted to o and p-bromoanisoles, 1-methoxy-4-bromonaphthalene and 2-bromothiophene respectively. Regiospecificity of the enzymatic bromination of anisole was tested and found to be the same as in the chemical reaction with NaOBr. The enzyme also acted on substituted alkenes such as styrene, cyclohexene, trans-cinnamic acid, trans-cinnamyl alcohol and cis-propenylphosphonic acid, to give the respective bromohydrin compounds or decarboxylated bromo compound. These bromohydrin compounds were always mixtures of stereoisomers. In the light of the above findings together with the previous studies concerning the halogenation mechanism, the bromoperoxidase of C. pilulifera was considered to have no specific restriction site for these substrates.     

Expression of the vanadium-dependent bromoperoxidase gene from a marine macro-alga Corallina pilulifera in Saccharomyces cerevisiae and characterization of the recombinant enzyme

The vanadium-dependent bromoperoxidase from the marine macro-alga Corallina pilulifera was heterologously expressed in Saccharomyces cerevisiae. The enzyme was purified and crystals in "tear drop" form were obtained. The catalytic properties of the recombinant enzyme were studied and compared with those of the native enzyme purified from C. pilulifera. Differences in thermal stability and chloroperoxidase activity were observed. The recombinant enzyme retained full activity after preincubation at 65 degrees C for 20 min, but the native enzyme was completely inactivated under the same conditions. The chlorinating activity of the native enzyme was more than ten times higher than that of the recombinant enzyme. Other properties, such as K(m) values for KBr and H(2)O(2), and optimal temperature and pH, were similar for each source of C. pilulifera bromoperoxidase.     

A comparison of the north-and southe european associations of Corallina officinalis L.

A comparison betweenCorallina officinalis associations from the northern Adriatic and the Icelandic coastal area is given regarding their floristic composition, zonal position and physionomy, as related to the environmental parameters and the different origins of the respective floras.     

珊瑚藻R-藻红蛋白rpeA和rpeB基因全长cDNA克隆与序列分析(英文)

依据珊瑚藻 (CorallinaofficinalisL .)藻红蛋白rpeA和rpeB的DNA序列 (AF5 1 0 986 )设计引物 ,通过PCR RACE方法扩增得到rpeA和rpeB的cDNA序列 .序列分析表明 ,该序列采用多顺反子转录策略 ,全长 2 2 5 7bp(AF5 42 5 5 4) ,排布顺序为 5′UTR rpeB 间隔区 rpeA 3′UTR .5′非编码区 4 93bp ,rpeB基因 5 34bp ,基因间隔区 1 0 1bp ,rpeA基因 4 95bp ,3′非编码区 6 34bp .在rpeA和rpeB的基因起始密码子上游均存在类似原核核糖体结合的Shine Dalgarno (SD)序列 .在rpeA基因终止密码子下游 1 1 0bp处还存在着一个可能的开放阅读框架 .经检索GenBank发现 ,真核红藻藻红蛋白中尚无有关cDNA序列的报道     

SNPs reveal geographical population structure of Corallina officinalis (Corallinaceae,Rhodophyta)

We present the first population genetics study of the calcifying coralline alga and ecosystem engineer Corallina officinalis. Eleven novel SNP markers were developed and tested using Kompetitive Allele Specific PCR (KASP) genotyping to assess the population structure based on five sites around the NE Atlantic (Iceland, three UK sites and Spain), spanning a wide latitudinal range of the species’ distribution. We examined population genetic patterns over the region using discriminate analysis of principal components (DAPC). All populations showed significant genetic differentiation, with a marginally insignificant pattern of isolation by distance (IBD) identified. The Icelandic population was most isolated, but still had genotypes in common with the population in Spain. The SNP markers presented here provide useful tools to assess the population connectivity of C. officinalis. This study is amongst the first to use SNPs on macroalgae and represents a significant step towards understanding the population structure of a widespread, habitat-forming coralline alga in the NE Atlantic.     

Purification of bromoperoxidase from Pseudomonas aureofaciens.

A Bromoperoxidase has been isolated and purified from Pseudomonas aureofaciens ATCC 15926 mutant strain ACN. The purified enzyme was homogeneous as determined by polyacrylamide gel electrophoresis and ultracentrifugation. This bromoperoxidase can utilize bromide ions in the presence of hydrogen peroxide and a halogen acceptor for the catalytic formation of carbon-halogen bonds. The homogeneous enzyme also has peroxidase and catalase activity. Based on the results from gel filtration and ultracentrifugation, the molecular weight of this procaryotic bromoperoxidase is 155,000 to 158,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows a single band having the mobility of a 77,000-molecular-weight species. We thus conclude that this bromoperoxidase exists in solution as a dimeric species. The heme prosthetic group of bromoperoxidase is ferriprotoporphyrin IX. The spectral properties of the native and reduced enzyme are reported. This bromoperoxidase is the first halogenating enzyme purified from procaryotic sources.     

The dodecameric vanadium-dependent haloperoxidase from the marine algae Corallina officinalis: cloning, expression, and refolding of the recombinant enzyme

The dodecameric vanadium-dependent bromoperoxidase from Corallina officinalis has been cloned and over-expressed in Escherichia coli. However, the enzyme was found to be predominantly in the form of inclusion bodies. This protein presents a challenging target for refolding, both due to the size (768 kDa) and quaternary structure (12 × 64 kDa). Successful refolding conditions have been established which result in an increase in the final yield of active bromoperoxidase from 0.5 mg to 40 mg per litre of culture. The refolded protein has been characterised and compared to the native enzyme and was shown to be stable at temperatures of 80 °C, over a pH range 5.5–10 and in organic solvents such as ethanol, acetonitrile, methanol, and acetone. The novel refolding approach reported in this paper opens up the full potential of this versatile enzyme for use in large scale biotransformation studies.     

Purification and properties of bromoperoxidase from Pseudomonas pyrrocinia

A bromoperoxidase was purified and partially characterized from Pseudomonas pyrrocinia ATCC 15958, a bacterium that produces the antifungal antibiotic pyrrolnitrin. The purified enzyme preparation was homogeneous as determined by polyacrylamide gel electrophoresis and ultracentrifugation. The molecular mass of the enzyme was estimated to be 154 kDa +/- 3 kDa as determined by gel filtration and ultracentrifugation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed a single band with the mobility of a 76-kDa species. Therefore, in solution at neutral pH, bromoperoxidase exists as a dimeric species. The isoelectric point was 5.0. The prosthetic group of this procaryotic bromoperoxidase was ferriprotoporphyrin IX. The spectral properties of the native and reduced enzyme are reported. The purified enzyme showed brominating as well as peroxidase and catalase activity.     

Vanadium K-edge absorption spectrum of bromoperoxidase from Ascophyllum nodosum

With synchrotron radiation from the Bonn 2.5 GeV synchrotron, high-resolution absorption spectra have been measured at the vanadium K-edge of bromoperoxidase from the marine brown alga Ascophyllum nodosum and several model compounds. The near-edge structure (XANES) of these spectra was used to determine the charge state and the coordination geometry around the vanadium atom. For the active enzyme a coordination charge of 2.7 was found which is compatible with a formal valence of +5, assuming coordination by atoms with a high electronegativity such as oxygen or nitrogen. For the reduced enzyme the coordination charge value of 2.15 indicates the reduction of the valency by 1 unit. Our results suggest that the coordination sphere of the vanadium atom in the native enzyme consists of at least seven oxygen atoms in a distorted octahedral environment with an average bond length of about 2 A. Through the reduction process, the coordination sphere of the vanadium atom changes with a simultaneous decrease of the coordination cage. These results agree with those deduced from previous EPR and 51V-NMR measurements.