| Ginsenoside is the main active substance of Panax ginseng CA Mey,which has anti-tumor,regulates immunity,delays aging and other medicinal development and health care value,especially ginsenoside Compound K(CK),Rh2 and Rg3.It has a higher therapeutic effect in the treatment of diseases,but the ginseng saponin is extremely low in ginseng and lacks a wide and stable source.Therefore,how to obtain rare ginsenosides is one of the research hotspots in the field of scientific research.Since the saponins of ginsenosides are the same,but the number of glycosylation modifications is different,the ginsenosides containing less glycosyl modifications can be prepared by hydrolyzing a glycosyl group containing a large amount of ginsenosides modified with a polysaccharide.Bio-derived glycosidase can specifically hydrolyze glycosidic bonds in sugar-containing compounds such as glycosides or glycoproteins,which have the advantages of wide source,mild reaction conditions,high specificity,low by-product content and variety,and can be applied to bioconversion of ginsenosides.In this study,a large number of glycosidase BglSK(derived from Sanguibacter keddieii)and BglSp(derived from Sphingomonas sp.2F2)were prepared by prokaryotic expression,and optimize its reaction conditions.A system for converting a large amount of ginsenosides Rb1,Rb2,Rc and Rd into rare ginsenoside CK was constructed.The specific results are as follows:1.Prokaryotic expression of glycosidase BglSK and BglSpAfter codon optimization and gene synthesis of glycosidase BglSK and BglSp gene sequences,three different prokaryotic expression vectors,pET32a,pET14b and pCold-SUMO,were constructed,and the prokaryotic expression vector pET14b-BglSKand pCold-SUMO-BglSp with the best soluble expression was screened.2.Enzymatic activity verification of glycosidase BglSK and BglSpThe glycosidase hydrolysis specificity was identified using a large amount of ginsenoside monomers Rb1,Rb2,Rc and Rd as substrates.Identification of glycosidase BglSK has C-3 exo-and endo-cutting activity,and C-20 exo-cleavage activity,its hydrolysis pathway is:Rb1?GypXVII?GypLXXV?CK,Rb2?CO?CY,Rc?C-Mc1?C-Me,Rd?F2?CK;glycosidase BglSp has C-3 exo-activity and C-20 exo-activity,and the hydrolysis pathway is:Rb1?GypXVII?F2,Rb2?CO?F2,Rc->C-Mc1?F2,Rd?F2.3.Optimization of induction conditions for prokaiyotic expression of glycosidase BglSK and BglSpThe optimal induction expression conditions of glycosidase BglSK and BglSp were 15?.0.1 mM IPTG concentration,and 10.77 mg glycosidase BglSK and 7.88 mg glycosidase BglSp were purified using AKTA protein purification system.4.Study on the properties of glycosidase BglSK and BglSpThe optimum reaction temperature range of glycosidase BglSK and BglSp is 35-40?,and the optimum reaction pH range is 7.5-8.5.The effects of different metal ions concentration on enzyme activity were detected,and it was found that when the ion concentration was higher than 10 mM,will inhibit the enzyme reactivity.5.Catalytic pathway analysis of glycosidase BglSK and BglSpThe glycosidase hydrolysis specificity was identified using a large amount of ginsenoside monomers Rb1,Rb2,Rc and Rd as substrates.Glycosidase BglSK hydrolysis pathway:Rb1?GypXVII?GypLXXV?CK,Rb2?C-O?C-Y,Rc?C-MC1?C-MC,Rd?F2?CK;glycosidase BglSp hydrolysis pathway:Rb1?GypXVII?F2,Rb2?C-O?F2,Rc?C-MC1?F2,Rd?F2.6.Establish a system in which glycosidase BglSK and BglSp transform ginseng total saponins into CKA large number of ginsenosides in ginseng total saponins were converted into rare ginsenosides CK by Two methods of glycosidase BglSK single enzyme conversion combined with glycosidase BglSK and BglSp double enzymes.When a glycosidase BglSK single enzyme was transformed,a large amount of ginsenosides(Rb1,Rb2,Rc and Rd)were converted.The ratio was 65.64%.When the two enzymes were combined,the molar conversion of a large amount of ginsenoside(Rb1,Rb2,Rc and Rd)was as high as 88.00%,which was 22.36%higher than that of the single enzyme conversion. |
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