Purification, Properties And Immobilization Of Peroxidase From Lotus Root (Nelumbo Nucifera Gaertn.)

Peroxidase (POD,EC1.11.1.7) widely exists in all kinds of animals, plants and microorganisms, and its structure and action mechanism because of different origin, but is not identical. It belongs to the oxidase of containing heme, generally containing metals such as Fe²⁺, Cu²⁺. Peroxidase can oxidize a variety of substrates mainly through catalyzing H₂O₂ or other peroxide. It is widely used in proteins, peptides, hormones, biodegradation, nervous system as well as the study of biomedical detection, sewage treatment, biosynthesis, food processing and biosensors.Lotus root (Nelumbo nucifera Gaertn.), the perennial aquatic herb of the family Nymphaeaceae, is our country main aquatic vegetables, supplying edible rhizome. It includes carbohydrate, protein, vitamin, mineral substance, and its flavor is unique. Lotus root can grow up in many places of China, having two function of nutrition and medicine. It has good health care function, and is a kind of food for eating and medicine.In this paper, purification, properties, fuction group and Immobilization of POD were studied. The experiment not noly provided the theory basis for expanding the origin of POD and further studies, but also provided the theory support for Lotus root full development and maintaining freshness. The findings are as follows:1. Purification of peroxidase from Lotus rootPeroxidase from the fresh Lotus root was purified using tissues mashing, buffer solution extraction, ammonium sulfate precipitation, ion exchange chromatography on DEAE-Sepharose Fast Flow column and gel filtration chromatography on Sephacryl S-200. The purification fold was 39.77, and the recovery was 11.9%. 2. Properties of peroxidase from Lotus rootThe molecular weight of Peroxidase from Lotus root is approximately 41.5 KD. The optimum pH and optimum temperature for the enzyme was pH 5.0 and 35℃. Stable conditions for peroxidase activity were pH6.0～pH7.0 and 35℃～50℃. The apparent Km values of the enzyme with different concentration of H₂O₂ as the substrate was 9 mmol/L at 25℃and pH 7.2. The enzymatic reaction initial velocity,V₀,was 2.50μmol/(L·min).Oxalic acid can activate POD activity. DEAE, KSCN and carbamide had no effect on POD cativity. SDS and NaCl partially inhibited POD activity, and furthermore, ASA inhibited POD activity completely. Cu²⁺ and Zn²⁺ enhanced POD activity. Ba²⁺ and Mg²⁺ didn't effect POD activity obviously. Na⁺, Li⁺ and K⁺ partially inhibited POD activity. Mn²⁺ and Fe²⁺ inhibited POD activity seriously. Methanol, ethnanol, glycol and ipropylalcohol can all inhibit POD activity. Methanol and ethnanol inhibited the activity obviously, ipropylalcohol is nest ,and glycol had slight relatively effect on POD activity.3. function group of peroxidase from Lotus rootThe study of function group of peroxidase from Lotus root indicated Ser, Sulfhydryl, disulfid bond and Met residues were essential function groups of POD. A few of His and Arg residues were related to POD activity. Lys and Tyr residues had no effect on POD activity.4. Immobilization of peroxidase from Lotus rootPOD was immobilized by sodium alginate and glutaradehyde. The experiment made primary study to the condition of POD immobilization and the properties of immobilized enzyme. The results indicated that when the sodium alginate concentration was 4% , calcium chlorideon concentration was 2%, glutaradehyde concentration was 0.25%, and immobilized time was 60 min , the Immobilization effect was best. The optimum pH and optimum temperature for immobilized enzyme was pH 5.0 and 55℃. The relative activity of immobilized enzyme remained 80% after it had been used 8 times.