Purification And Characterization Of Rutin Degrading Enzymes From Tartary Buckwheat, And Screening Of RDEs-producing Strain

Fagopyrum tatarican(L.)Gaerth belongs to dicotyledonous plant and also acts as cereal crop.It not only contains rich rutin,but also exists some rutin degrading enzymes in tartary buckwheat seeds.Under certain condition,the rutin degrading enzymes can hydrolyze rutin to produce quercetin,rutinose,et al.Quercetin has few physiological functions such as antioxidative,radical scavenging activity and anticancer,consequently,it has high medicinal value and provides a good perspective on commercial market.At present,there is only one way that acid hydrolyze rutin to produce quercetin.However, acid may easily lead to environmental pollution.The technique of microorganism fermentation that has advantages of low-cost and less pollution,it has been widely using in modern medicinal industry.This paper mainly concerns the purification and characterization of rutin degrading enzymes from tartary buckwheat,and screening of RDEs-producing strain.Meanwhile, this strain has been identified from the level of morphology,biochemical physiology and 18s rDNA-ITS.Experiment condition has been optimized to improve the contents of quercetin.It provides an effective way to use microorganism fermentation assisted extraction of quercetin.The result obtained as follows:1.Rutin degrading crude enzymes was extracted from tartary buckwheat seeds.And then it was purified by using Sephadex G-100 column and DEAE cellulose-32 anion exchange column.The specific activity of rutin degrading enzymes was increased from 59.2 U/mg to 671.4 U/mg.The yield and purified fold are 3.5%and 11.3,respectively.2.The molecular weight of the rutin degrading enzymes was about 60 KDa which was detected by the SDS- polyacrylamide gel electropheresis.3.The optimum reaction condition of the rutin degrading enzymes were 40℃,and pH 5.0 by ultraviolet spectrophotometry. 4.Three RDEs-producing strains were isolated from fusty tartary buckwheat flour, their code was W₂,Y₂ and B₃.Among these three strains,B₃ had the highest activity,which was as high as 3.42U/mL,and 171.0U.5.B₃ was identified as Penicillium farinosum from the research of its shape, physiological characters and 18S rDNA-ITS.B₃ colony is blue and particle,it was found that there is a septum in hypha.The top of sporophore is not intumescence and vesicle. Several branching form lots of symmetrical or unsymmetrical sterigma.The whole strain is composed of sporophore,sterigma and spore,which looks like a broom.Then analysis of 18S rDNA-ITS gene of B₃,it was found that the sequence homology is as similar as Penicillium farinosum,and achieve to 100%in the phylogenetic tree constructed.6.B₃ strain inoculated the tartary buckwheat flour at 30℃,then extracted using 80% ethanol.The yields of quercetin was improved to 5.1 fold compared to the control.