Purification And Immobilization Study Of Jerusalem Artichoke Leaves Peroxidase

Jerusalem artichoke, tuberous-rooted perennial (Helianthus tuberosus) of the family Asteraceae (aster family), is native to North America and is also cultivated in the North and South of China. It is of wide viability, resistant to cold, aridity, leanness and sand. The tubers grow underground, gnarly and uneven. The tubers store the carbohydrate inulin. For that reason, they are an important source of fructose for industry. Most researches study on inulin production from the tubers. The stems and leaves are always brushed off, resulting large waste. Sometimes the stems and leaves are used for stock feed with a low economic additional value. There are no reports about peroxidase extraction from Jerusalem artichoke leaves.Peroxidase (POD, donor: H₂O)2, oxido-reductase, EC 1,11,1,7), with its stable chemical and physical properties, is a kind of glycoprotein containing a heme, usually feriprotoporphyrin IX. As the marker enzyme, it is widely used in medical diagnose, the chemical of immune cytohistology as well as the study of protein, polypeptide, hormone, virus, neuraxon transfer, the food and sanination detection, the treatment of the three wastes, the syntheses of macromolecule materials and the degradation of lignin. Taking its specific stability and fine reaction performance into consideration, it is even supposed that the peroxidase could hold potential application in the catalyse synthese of polyhydroxybenzene and aromatic amine materials someday. The enzyme-catalysed polyhydroxybenzene is free of formaldehyde and owns huge potential in photoelectronical materials due to itsл-лconjugate structure. Currently, the POD is abstracted from horseradish (HRP) home and abroad. The POD used in China is mainly imported.The extraction and purification study of Jerusalem artichoke POD (HLP), as well as its chemical and physical properties research provides the references for the exploitation of Jerusalem articoke leaves. The immobilization of HLP and the application for glucosuria test paper lay a theoretical foudation for the utilization of Jerusalem artichoke resources.1. Fesibility of abstraction and purification of Jerusalem artichoke From the comparison of enzyme extraction liquid of Jerusalem artichoke leaves, bamboo shoot shields, sugar cane leaves and bean shells, the order of enzyme activities is as follow: sugar cane leaves(6.401×10³U/g)> bamboo shoot shields(5.859×10³U/g)> Jerusalem artichoke leaves(5.838×10³U/g)> bean shells(4.038×10³U/g). PAGE shows that HLP differs from HRP, in view of molecular weight and electric charge, belonging to acid enzyme, thus HLP could be a new kind of POD resource. It is found that the POD activity reaches the highest level in anthesis, compared with the seedlings and mature plants and the POD activities in different parts vary, the middle part leaves being the highest, then the leaves near the roots, the top leaves being the lowest enzyme activity. PAGE tells that the enzyme activities are lower in the young and old leaves, which could be related with the synthesis and degradation of POD. As a whole, it is possible to collect the mid part leaves and the leaves near the roots for the use of POD extraction and purification.2. Extraction, Ammonium Sulfate Fractionation and Purification of HLP A peroxidase from Jerusalem artichoke was pufiried by using pH6.8, 0.1mol/L Na2HPO4-NaH2PO4 buffer for the extraction, 50%-90% ammonium sulfate fractionation and Sephadex G-75 filtration and the activity recovery was 22.4%, 16.2 times purified then the enzyme extracion. After DEAE-Sepharose Fast Flow filtration, 3 bands were stained when POD isoenzyme electrophoresis indicating that HLP consisting of 3 isoenzymes.3.Nature of Chromatographically pure HLP Determined by Sephadex G-75, the moleculare of HLP is approximately 23600. Spectroscopy analysis showed a typical Soret band at 320nm. Emission spectrum indicated 2 clear emission peak at 310nm and 510nm. The optimum pH was 5.0 and the optimun temperature was 40℃. HLP is stable to heat. Keeping...