Purification, Some Properties And Modification Of Groups And Immobilization Of The Sucrase From Mentha Haplocalyx Briq 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 assimilation and transportion, 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.Mentha haplocalyx Briq belongs to Menthinae Briq family of Labiatae Linn. They are aromatic, annual or perennial herbaceous plants with its leaves the mainly fruits. There are 12 categories of Mentha haplocalyx Briq in our country, and it is one of the most cash crops in northern hemisphere. With more and more researches on phytochemistry and pharmacology, Mentha haplocalyx Briq has been widely used in food processing food industry and medical area. This paper can fill the domestic gap of the related research on Mentha haplocalyx Briq through a theoretical foundation. In addition, the paper will make more people know the value of the Mentha haplocalyx Briq, and develop their utilization values so as to enhance the economic values.The results of our study were followed:1. Purification of sucrase from Mentha haplocalyx Briq leavesCrude enzyme solutions were obtained from Mentha haplocalyx Briq leavesby tissues mashing,ultrafiltration and sequential ammonium sulpate precipitation. And then Sucrase was purified through the DEAE-Sepharose Ion-exchange chromatography and Sephacryl S-200 gel filtration chromatography. The purified sucrase was shown to be homogenous on SDS-PAGE and IEF eletrophoresis. The specific activity was 67.06U/mg,and purification fold was 186.3. The recovery of sucrase activity was 24.9%.2. Properties of sucraseThe molecular weight of the enzyme is 130.0KD and subunit is 64.5KD as assay by Sephacryl S-200 and SDS-PAGE method. Isoelectric point of the sucrase is 5.6 showed by IEF-PAGE.The kinetic characters of the enzyme have been studied. The optimum pH and optimum temperature for the enzyme are pH 5.0 and 55℃. Km is 12.25mmol/L.3. Inhibitors of sucraseAmong several mental ions, As⁵⁺,Ag⁺,Pb²⁺ are strongly inhibited the activity, Cr³⁺,Mg²⁺,K⁺,Ba²⁺,Fe²⁺ have little effect on sucrase, but Cu²⁺,Mn²⁺ can activate the enzyme activity. With chemical reagents, EDTA were the strongest inhibitors, followed by H₂O₂,urea,Vc.4. Chemical modification of sucraseSucrase was selectively modified by NBS,PMSF,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 sucrase activity.5. Immobilization of sucraseSucrase was immobilized by means of PVA-CA-glutaraldehyde embedding method. So in this paper, immobilization of sucrase lipase was studied. The optimum immobilization conditions is 3%CA,the ratio of carrier to enzyme liquid being 1:2.5(V/V),0.5% glutaraldehyde the time of immobilization being 0.5h, the recovered sucrase activity was 45.7%. The optimum pH and optimum temperature for the Immobilization of sucrase are pH 5.0 and 55℃,the same as dissociate enzyme and also have wider range fit for reaction.The result showed that the immobilized sucrase activity with no loss deposited 4℃refrigerator one month.