Preparation Of A Series Of Rare Pro-saikosaponins By Enzymatic Hydrolysis

Saikosaponin is main bioactive chemical constituents of Bupleurum chinense.The content of saikosaponin A（SSA）,saikosaponin B₁（SSB₁）,saikosaponin B₂（SSB₂）and saikosaponin D（SSD）was higher than other pentacyclic triterpenoids in the herb.However,due to their large molecular weight and high polarity,these primary glycosides are poorly absorbed in human small intestine.After they are transformed into pro-saikosaponin or saikogenin,these resulting substances can be better absorbed then,so as to play a better role in their efficacy.The methods to prepare saikosaponin include conventional acid hydrolysis,microbial transformation and enzymatic hydrolysis.The acidic wastewater produced by the acid hydrolysis will cause serious damage to environment,and saikosaponin is usually further hydrolyzed to produce saikogenin and other by-products.The fermentation process during microbial transformation is time-consuming and difficult to control,and the operation is tedious.However,the enzymatic hydrolysis has the merits of mild reaction conditions,strong specificity and high efficiency,and it has been widely used in the conversion of a variety of saponins.In this research,I constructed a key technology of enzymatic hydrolysis and optimized conditions to obtain a series of rare pro-saikosaponins.In addition,aβ-D-glucosidase was recombinated and its enzymatic properties were characterized,which laid a solid foundation for efficient hydrolysis of saikosaponin by reusable immobilized enzyme in further study.This research includes the following three main sessions:1.Study on the enzymatic hydrolysis of SSB₁and SSB₂to obtain pro-saikosaponin A（PSA）and pro-saikosaponin D（PSD）.Firstly,HPLC-UV based method were established for the content determination of SSB₁,SSB₂,PSA and PSD;then,the conversion ratio of primary glycosides was used as the index for the comparison of the conversion effect ofβ-glucanase,β-glucosidase,cellulase,naringinase or snailase on SSB₁/SSB₂.The proper enzyme was selected,and then single factor and response surface methodology（RSM）were employed to optimize conditions of enzymatic hydrolysis.The established technology was applied to obtain PSA and PSD,and the products were purified by preparative HPLC and their chemical structures were identified by LC-MS,¹H-NMR and ¹³C-NMR.The results showed that cellulase was the most proper enzyme for hydrolysis of SSB₂.While enzyme/substrate ratio was 80:1（mg:mg,w/w）,the hydrolysis duration was 33 hrs,the reaction temperature was 60°C,and the buffer p H was 4.7,the highest conversion ratio was 95.0%.On the other hand,snailase was the most proper enzyme for hydrolysis of SSB₁.While enzyme/substrate ratio was 30:1（mg:mg,w/w）,the hydrolysis duration was 33 hrs,the reaction temperature was 56°C and the buffer p H was 4.5,the highest conversion ratio of SSB₁was achieved at 99.1%.2.Study on enzymatic hydrolysis of SSA and SSD to obtain pro-saikosaponin F（PSF）and pro-saikosaponin G（PSG）.Firstly,HPLC-UV based method were established for the content determination of SSA,SSD,PSF and PSG;Then,the conversion ratio of SSA/SSD was used as the index,and single factor and response surface methodology（RSM）were employed to optimize conditions of enzymatic hydrolysis.The established technology was applied to obtain PSF and PSG,and the products were purified by preparative HPLC and their chemical structures were identified by LC-MS,¹H-NMR and ¹³C-NMR.while enzyme/SSA ratio was 44:1（mg:mg,w/w）,the hydrolysis duration was 12 hrs,the reaction temperature was 39°C,and the p H was 6.0,SSA could be completely transformed into PSF;On the other hand,while enzyme/SSD was 70:1（mg:mg,w/w）,the hydrolysis duration was 14 hrs,the reaction temperature was 37°C and the buffer p H was 5.5,the highest conversion ratio of SSD was achieved at 99.2%.3.Recombinant and characterization ofβ-D-glucosidase.The expression vector p PIC9K-Gla was synthesized,and the vector was electroporation into the P.pastoris GS115 to obtain a high resistant to G418.The fermentation conditions were then optimized.The results showed that the enzyme activity was the highest after induction for 6 days when p H of medium was 6.0 and the amount of methanol was 1.5%（v/v）.The expressed enzyme showed best performance at 50°C when the p H of the buffer was 6.0.1 m M K⁺and 5 m M Fe³⁺had slight activation on the enzyme activity,whereas Cu²⁺and 1 m M Fe³⁺had no effect.Meanwhile Mg²⁺or SDS significantly inhibited the activity of recombinant enzyme,and Tween-80 andβ-ME exhibited slightly inhibition effect.Theβ-D-glucosidase had a good resistance to 10%methanol,and its activity still maintained more than 83%.The expressed enzyme also has a high resistance to glucose,its activity is less affected by the feedback inhibition of the product.When p NPG was used as substrate,the Michaelis constant K_mof recombinantβ-D-glucosidase was 2.43 m M,and the maximum reaction rate V_maxwas3.71μM/min.The substrate specificity study showed that the enzyme could hydrolyze the glycosidic bond at the end of saikosaponins including SSA,SSB₁,SSB₂and SSD.