Seperation, Purification And CDNA Cloning Of Peroxidase From Longan(Dimocarpus Longan Lour) Pericarp

Peroxidase (POD) is an enzyme which plays a very important role in fruit browning. In our knowledge, the studies on these PODs are focused on physiology and biochemistry. We study on the POD purification, mass spectrum analysis and cDNA cloning in longan pericarp.The crude enzyme extraction was fractioned with 40% saturation solid ammonium sulfate, separated by Streamline Phenyl column, DEAE-Cellulose column, Phenyl Sepharose High Performance column and Superdex 200 prep grade column, respectively. The enzyme was detennined to be homogenous by SDS-PAGE which apparent molecular weight is about 48KD. The precise molecular weight determination showed the precise molecular weight is 45.65KD. Compared with gel filtration standard showed that the relative molecular weight of POD is about 45KD as well, which indicate that the purified POD is monomer. Isoelectric focusing of POD reveals two isozyme activities A and B, with different pIs which belong to anionic POD. It's difficult to separate them because of their same molecular weight and characteristics.Matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF MS) was applied to the analysis of peptide mass fingerprinting (PMF) in POD A. The PMF data of POD A and was used in Mascot search, but no data matched. Then, the technique of electric spray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF2 MS) was used in POD A identification. Two tryptic-digested peptide fragments were sequenced by ESI-MS/MS, which sequences were: WFLSGGDWYSLF and VRESAWLDNDADLLAVP. No data matched in database search as well. Multiple alignments with other POD sequences showed there are some conserved amino acids in these sequences.Total RNA of longan pericarp was extracted, and conserved region of POD cDNA was obtained by degenerated oligonucleotide primed RT-PCR. After that, the rapid amplification of cDNA ends (RACE) was performed to the 3' and 5' direction, and full length cDNA encoding POD was obtained from longan pericarp. The full sequence is 1290bp in length, containing a 996 bp-nucleotide of open reading frame (ORF), encoding a putative protein of 332 amino acid residues. When aligned with primary structure of BP1 of Barley, HRP C of horseradish, and SBP of soybean, active site, substrate binding pocket, heme binding site and calcium binding sites were found in the deduced amino acid sequence. The results also showed that some amino acid peptides got by ESI tandem MS...