Purification And Some Properties Of Sucrase From Tobacco(Nicotiana Tobacum. L) Leaves

Sucrase (EC,3.1.2.26) is widespread in prokaryotes and eukaryotes. Sucrase catalyzes the irreversible hydrolysis of sucrose into glucose and fructose, the mainforms of carbon and energy supplies in plant growth and development. Sucrase plays an important role in transforming the photosynthetic products from the source tissue to the sink tissue.In plants, due to the central role of sucrase in assimilate transport, which is considered as a key enzyme for carbohydrate metabolism. Moreover, sucrase also plays important functions in controlling of cell differentiation and cell enlargement, signal transduction, and plant response to wounding, infection and kind of stress.Fire-cured tobacco is one of the important cash crops in tobacco-growing area in china, and a main source of raw material for tobacco manufacturing. Over the past few years, compared with smoking and health, quality control for cigarette product has been an increased interest in global.Sugars are inevitable constituents in all types of tobacco material, and both their quantities and proportions strongly affect not only the quality of tobacco as a raw material, but the properties of the final tobacco products as well. Sugars are frequently added to tobacco during the manufacturing process, being generally recognized as safe tobacco additives, are usually reported to serve as flavour and humectant. Sugars like sucrose, glucose and fructose are the materials basis for synthesis of organic acid, cariotenoids, and other nutrient and aromatic ingredients. Therefore reliable determination of these compounds in tobacco appears to be essential for the classification of tobacco material according to its content.The results of our study were followed: 1. Purification of invertase from tobacco leavesCrude enzyme solutions were obtained from tobacco leaves by tissues mashing,ultrafiltration and sequential ammonium sulpate precipitation. And then invertase was purified by DEAE-Sep-harose Ion-exchange chromatography and Sephacryl S-200 gel filtration chromatography. The purified sucrase was characterized by SDS-PAGE. The specific activity was 65U/mg, purification fold was 216. The activity recovery of sucrase was 17.4%.2. Properties of sucraseThe molecular weight of the enzyme is 129kd and subunit is 63.5kd as assay by Sephacryl S-200 and SDS-PAGE. The kinetic characters of the enzyme have been studied. The optimum pH and optimum temperature for the enzyme are pH 4.0 and 45℃. Km is 13mmol/L and the Vmax is 7.67μg/min.3. Inhibitors of sucraseAmong several mental ions, Hg²⁺,Pb²⁺,Ag⁺ and EDTA are strongly inhibited the activity, Cr³⁺,As⁵⁺ have little effect on sucrase, Mg²⁺,K⁺ have no effect on it, but Mn²⁺,Cu²⁺ can activates the enzyme activity.4. Chemical modification of sucraseSucrase was selectively modified by PMSF,NBS,IAc,BrAc,SUAN,TNBS,PCMB,DTT. The results show that PMSF,NBS,PCMB inhibited sucrase activity strongly ,while BrAc,IAc,TNBS,SUAN,DTT have little effect on invertase activity.