Isolation And Purification And Gene Cloning Of Pulsatilla Saponin Carbohydrate Hydrolysis Enzyme Type ?

Pulsatilla saponin carbohydrate hydrolase enzyme could specifically hydrolyze the glycosidic bond on Pulsatilla saponin,and converted polysaccharide-based Pulsatilla saponin into low-glycosylated Pulsatilla saponin,which could not only improve the bioavailability of Pulsatilla saponins,but also increased its pharmacodynamic value.In this thesis,the separation and purification of Pulsatilla saponin carbohydrate hydrolysate type II enzyme produced by fermentation of Absidia sp.P00 r were studied.In liquid fermentation culture study of Absidia sp.P00 r,it was found that Pulsatilla saponin glycosyl hydrolyzation type II enzyme produced larger yields when cultured in 5° wort medium prepared with 70% Pulsatilla chinensis extract as inducer for 120 h.The effect was good.The DEAE-Cellulose(DE52)anion exchange column was used to separate and purify.The electrophoretically pure Pulsatilla saponin glycosyl hydrolyzing type II enzyme protein was obtained.After the protein was transferred to the PVDF membrane by electroporation,the N-terminal sequence of the protein was determined.The N-terminal sequence of this enzyme was determined to be YPDSVQHAETVQNLI.Based on the measured N-terminal sequence of Pulsatilla saponin glycosyl-hydrolyzing type II enzyme protein and the measured peptide mass spectral data,the NCBI website and the Matrixscience website's protein database were compared for homology,and degenerate primers were designed based on homology.Designed degenerate primers based on the N-terminal sequence of Pulsatilla saponin glycohydrolyase type II enzyme,mRNA of Absidia sp.P00 r was used as a template for RT-PCR gene sequence retrieval,according to 3'RACE and 5'RACE techniques The gene sequence of the 3?-end and 5?-end of Type II enzyme was hydrolyzed by saponin-based saponin-based saponin,and the full-length sequence of the glycosyl hydrolase II enzyme of Pulsatilla saponin was obtained.Finally,the sequence of the extracted gene sequence was analyzed.Pulsatilla saponin glycosyl hydrolysis type II enzyme full-length 1376 bp,of which 1~84 bp was 5'UTR,a length of 84 bp;85~1137 bp was the CDS region of the gene,the length was 1053 bp;could encode 351 amino acids;1138~1365 bp was 3'UTR,length was 228 bp;1366~1376 bp was Poly A part,length was 11 bp.