The Purification Of Peroxidase From Chinese Kale And Expression In Escherichia Coli

Peroxidases (POD.EC1.11.1.x) are important oxidoreductases, which can effectively catalyze a series of organic and inorganic substances. Plant POD (EC1.11.1.7) not only play an important physiological role in the plant life cycle, but also be widely used in biotechnology. The properties of POD in Chinese kale, the purification of isoperoxidases and the expression of Bj-APX in the Escherichia coli BL21 were investigated in this study.Using guaiacol as substrate, the maximum activity of POD was determined at 55°C and pH 5.6. The thermostability of peroxidase was good. Trehalose, calcium chloride, magnesium sulfate and gelatin, with the concentration of 1g/L, can improve the stability of POD. Especially, the effect of calcium chloride and gelatin were obvious, and the activity of POD was improved 25% and 23% respectively compared with control.The zymogram of isoperoxidases showed that there were four isozymes in the young seedlings, whereas six isozymes appeared in the mature leaves. The treatments of plant regulator did not change the number of the isozymes compared with the control. However, ethephon, 2,4-dichlorophenoxy(2,4-D) and 1-naphthlcetic acid(NAA) improved the activity of POD, inversely KT did not. The purification of the isozymes was carried out by ammonia sulphate precipitation, ion-exchanged chromatography and preparative PAGE. Two isozymes, Bj1 and Bj4, were purified. The molecular weight of Bj1 and Bj4 was 66.2 and 32 KDa respectively on SDS-PAGE.Based on the conservative gene sequence of the POD in the plant belonging to the same genera with kale, which were published in the database, primer was designed and PCR amplification was carried out with kale's cDNA for template, 1200 bp and 500 bp DNA bands were obtained. The result of sequencing showed that they were highly conserved with matrix peroxidase gene SAPX and APX existed in the green vegetables and Arabidopsis, respectively. Two isozyme genes (Bj-SAPX and Bj-APX) were amplified accoding to conserved region of Brassica species. The sequences analysis showed that the similarity of Bj-SAPX and Bj-APX with Brassica oleracea BO-sAPX and Brassica oleracea BO-APX1 were 98% and 99% respectively. The Bj-APX gene was cloned into expression vector pET28a, transformed into E.coli BL21.The effect of induction condition on Bj-APX expression was investigated, including induction temperature, induction time and IPTG concentration.The optimum induction conditions were determined to be 30°C, 4h and 0.05mM IPTG. SDS-PAGE analysis showed that the molecular mass of recombinant APX was about 29 KDa. Hemin, calcium chloride and acetone can improve the permeability of E.coli BL21 which lead to the addition of hemin into the protein and higher enzyme activity. The optimal concentration was calcium chloride 3 g/L, acetone 7.5%, respectively.