Purification and biochemical properties of peroxidase isoforms from papaya (Carica papaya, Tainung No.2) fruit peel

• Main Authors: Min-ta Tsai, 蔡旻達
• Other Authors: Chen-Tien Chang
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/12217035530517280721

碩士 === 靜宜大學 === 食品營養研究所 === 98 === Abstract Two isoforms of peroxidase, POX-A and POX-B, were purified from ripe papaya (Carica papaya Tainung No. 2) fruit peel using sequential aqueous two-phase system extraction,Sephacryl S-100 HR gel filtration and preparative isoelectric focusing electrophoresis in the rotofor cell. The molecular masses of POX-A and POX-B were 45.2 and 32.7 kDa，respectively, as estimated by gelfiltration,and the isoelectric points were 3.81 and 8.72, respectively, as estimated by isoelectric focusing electrophoresis.Incubation of the purified peroxidase isoforms for 5-60 min at temperatures ranging from 30 to 80℃ revealed that the POX-A was stable from 30 to 40℃ whereas pox-B was stable from 30 to 50℃. At temperatures greater than 60℃, both isoforms were significantly inactivated. The rate of thermal inactivated of both isoforms does not reflect denaturation proceeding according to first-order kinetics. Heavy metal ion Cu2+ at a concentration of 4.55 mM and chemical modification reagent N-bromosuccinimide at a concentration of 0.9 mM significantly inhibited both POX-A and POX-B activities whereas chelating agent EDTA at a concentration of 4.5 mM and chemical modification reagents diethyl at a pyrocarbonate concentration of 4.5 mM, only significantly inhibited POX-B activity. Both isoforms oxidized 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid ; ABTS), catechol, ( o-phenylenediamine ; OPD), o-dianisidine and pyrogallol. The optimum pHs of POX-A and POX-B for varions hydrogen-donor substrates were almost the same,except for ABTS；pH 5.5 for POX-A and pH 4.0 for POX-B. Under optimal reaction conditions for each hydrogen-donor substrate, the best substrates for POX-A were ABTS, catechol and OPD whereas the best substrates for POX-B were ABTS and catechol.