Study Of Vanadium-dependent Bromoperoxidase Gene(vBPO) Phylogenetics Analysis, Prokaryotic Expression And Enzyme Activity Of Saccharina Japonica

The ocean contains a lot of bioactive halogen containing organic compounds,including halogenated hydrocarbons, halogenated phenols, halogenated tyrosine,halogenated fatty acids and halogenated terpenoids. Most of these organisms havepharmacological activity. Halogenated organic compounds are mainly derived frommarine algae biosynthesis. Therefore, to study the halide mechanism in the algae hascertain feasibility and high research value.Biological process mainly depends on the role of halogenated enzyme halide andhalogen enzymes including halogenated peroxidase and flavin dependent halogenatedenzyme. The vanadium dependent halogenated peroxidase (vHPO) is one of the moststudied kind among that. It can be divided into vanadium-dependent chlorinatedperoxidase (vCPO), vanadium-dependent bromine peroxidase (vBPO) andvanadium-dependent iodine generation peroxidase (vIPO) depending on its oxidationability of halogen ions. These three enzymes on the gene structure has the sameconservative metal ion binding sites, that is a histidine residues of imidazole ring, theregion for vanadium ion, amino acid series and acid phosphatase family (acidphosphatases) has a high degree of homology.Vanadium dependent halogen oxidase in red algae, green algae and brown algaehave been found, but vanadium dependent iodine peroxidase only were found in thebrown algae, especially some of the higher taxa, such as seaweed. Kelp is brownalgae common large economy in our country. The iodine content is higher, may withalgae body contains special halogen oxidase. This study constructed byhigh-throughput sequencing kelp transcriptome database and through the sequence, tobe obtained for the two kelp vanadium dependent iodine peroxidase genes, respectively, Sja vHPO1andSja vHPO2. The length of Sja vHPO1gene is2034bp,encoding677amino acids.The length of Sja vHPO2is1923bp, encoding640aminoacids. Through bioinformatics methods for these two genes in the gene structure andthe analysis of the physical and chemical properties, protein secondary structure and3D structure prediction and conservative comparative analysis.By transcriptome database retrieval, we got38amino acid sequence among30species vHPO genes.Then we built a phylogenetic tree combined with46vHPO genesdownloaded from the NCBI among37species and the amino acid sequence of thepap2acid phosphatase gene from three species.The red algae branch and.brown algaebranch can be completely separated, belonging to two major branches. The branch ofred algae also contains some photosynthetic bacteria and bacteria. According toendosymbiosis hypothesis, the earliest vBPO gene might come from thecyanobacteria species, consumed by eukaryotic host cells developed into modern redalgae. Most of the multiple copy genes appeared in the brown algae branch,suggesting that there might be a gene replication events, The brown algae vHPOhorizontal transfer of genes may come from red algae.In terms of the genetic time ofdifferent vHPO genes, vCPO came first, then evolved vBPO, and vIPO appeared atthe latest. Prokaryotic expression is a commonly used methods for protein expression.In this experiment, we the connected the two kelp vHPO genes into the pET-32-acarrier, then transfer it into E. coli BL21strain.We used IPTG as stimulus,thenpurified the two kinds of kelp protein Sja vHPO1and Sja vHPO2through the nickelcolumn method.Its concentration is0.17mg/ml and0.85mg/ml.To obtain protein enzyme activity test, we used the monochlorodimedonemethod which is supposed to be standard method to detecting the activity of vHPOproteins. High performance liquid chromatography (HPLC) method is accurate andconvenient method of liquid composition analysis in the biological and chemicalfields. We detecting the Sja vHPO protein catalytic activity by the HPLC analysis atthe conditions of25℃, pH6.5. The enzyme catalytic activity of iodine ion ions is2342.88U/mg and the bromine ions in the catalytic activity of5861.1U/mg. Explores the temperature and pH on the activity of the enzyme, determine the enzyme for.theprotein. The protein is Vanadium-dependent bromoperoxidase (vBPO).