Preparation For Minor Ginsenosides And Przewalskinic Acid A With High Physiological Activities By Enzyme Conversion

Ginseng and Salvia miltiorrhiza (Danshen) are the important traditional Chinese herbs. Ginseng has the efficacy of supplying energy, tonifying spleen, tranquilizing mind and promoting intelligence, etc; Danshen has the efficacy of promoting blood circulation, dispelling blood stasis, protecting heart and head blood-vessel, etc. Ginsenosides are the major physiological activity compositions of ginseng; Salvianolic acid B is one of the major water-soluble bioactive compositions of Danshen. The common characteristics of these natural products with multi-sugar moieties were low bioavail ability. After oral intake of herbs, these natural products were hydrolyzed by digestive enzymes and intestinal bacteria into the more active compounds that are easily absorbed by intestines. However, this kind of transformation in human body was very limited, and thus most of natural products were difficult to be absorbed and utilized, and were excreted. Therefore, the transformation of high-content natural products in vitro is of great value to many industries like Chinese medicine, health-care food and cosmetics.In this thesis, we systematically studied two ginsenosidase type I from Aspergillus niger g.848 and Aspergillus niger g.48 strain, examined their working mechanism and reaction kinetics in the hydrolization of glycosyls at 3-0-and 20-0-position in a number of Protopanaxadiol type ginsenosides. We further built up the preparation methods for producing highly-active minor ginsenosides, including C-K, F2, C-Y, C-Mc and C-Mx, through enzymatic transformation from low-cost PPD-type ginsenoside of American ginseng and Notoginseng leaves by crude enzyme from A. niger g.848 strain. We also studied enzymatic properties of Salvianolic acid-esterase from Aspergillus oryzae D30s strain and set up the strategy for preparing highly-active and small-molecular Danshensu and Przewalskinic acid A with this enzyme.Part I, the biotransformation of ginsenosides.In order to obtain minor ginsenoside C-K, F2, C-Y, C-Mc and C-Mx from PPD-type ginsenoside Rbl, Rb2, Rb3, Re and Rd, the following research was carried out in this part: â‘ Aspergillus niger g.848 and g.48 strain were fermented to product two kinds of ginsenosidases, respectively. â‘¡Then, two ginsenosidases type-I were isolated and purified. â‘¢The optimal reaction conditions of these two ginsenosidases were studied.â‘£Their working mechanism and enzymatic reaction kinetics were studied. ⑤Minor ginsenosides C-K, F2, C-Y, C-Mc and C-Mx were obtained from PPD-type ginsenosides of American ginseng and Notoginseng leaves ginsenosides using special ginsenosidase type-I from A. niger g.848 strain usingFirstly, Aspergillus niger g.848 and g.48 strain were fermented, then two kind of crude enzymes were precipitated by the method of ammonium sulphate precipitation. Two pure enzymes were isolated and purified from crude enzymes by DEAE-cellulose-DE52 anion exchange resin and active-PAGE technology. The molecular weights of these two enzymes were 75 kDa and 74 kDa, respectively. Both the enzymes can hydrolyze 3-O- position and 20-O- position multi-glycosly of PPD-type ginsenosides, but can not hydrolyze PPT-type ginsenosides. So, these two enzymes were classified to ginsenosidase type â…  The ginsenosidase type-â…  from A. niger g.848 strain was named G-I-g.848, and the ginsenosidase type-I from A. niger g.48 strain was named G-I-g.48, each. The optimal reaction temperature were both 45 ℃, the optimal pH were both 5.0.Enzymatic mechanism and enzymatic reaction kinetics of ginsenosidases G-I-g.848 and G-I-g.48 in hydrolyzing 3-O- and 20-O- glycosyl of PPD-type ginsenosides Rb1, Rb2, Rb3, Rc, Rd, F2 and 20(S)-Rg3 were studied, respectively. Ginsenosidases G-I-g.848 simultaneously hydrolyzed 3-O-Glc and 20-O- Xyl glycoside of ginsenoside Rb3 with two path way:one path way, hydrolyzed 3-O-Glc of Rb3â†'C-Mx1â†'C-Mxâ†'C-K (this is the main hydrolysis pathway); another path way, hydrolyzed 20-O-Xy1 of Rb3â†'Rdâ†'F2â†'C-K. However, the enzyme hydrolyzed ginsenoside Rb1, Rb2, Rc, Rd, F2 and 20(S)-Rg3 with only one path way:the enzyme firstly hydrolyzed 20-O-Glc of Rbl, then hydrolyzed 3-O-Glc, the paty way was Rb1â†'Rdâ†'F2â†'C-K; the enzyme firstly hydrolyzed 3-O-Glc of Rb2 or Rc, then hydrolyzed 20-O-Ara, the paty way were Rb2â†'C-Oâ†'C-Yâ†'C-K, Rcâ†'C-Mclâ†'C-Mcâ†'C-K, respectively. The enzyme hydrolyzed 3-O-Glc of Rd, F2,20(S)-Rg3 path way were Rdâ†'F2â†'C-K, F2â†'C-K,20(S)-Rg3â†'20(S)-Rh2â†'Ppdiol, respectively.The kinetic parameters of ginsenosidase type I from A. niger g.848 strain were showed as follow:while the enzyme hydrolyzes 3-O-Glc of Rb1â†'Gyp17, the Km= 16.6±1.6 mM, Vmax= 79.6±7.5 mM/h; while the enzyme hydrolyzes 3-O-Glc of Rb2â†'C-O,Km= 20.4±2.1 mM, Vmax= 45.6±4.62 mM/h; while the enzyme hydrolyzes 3-O-Glc of Rb3â†'C-Mxl, the Km= 15.1±1.4 mM, Vmax=10.8±1.1 mM/h; while the enzyme hydrolyzes 20-O-Xyl of Rb3â†'Rd, the Km=15.1±1.4 mM, Vmax=1.62±0.17 mM/h; while the enzyme hydrolyzes 3-O-Glc of Rcâ†'C-Mcl, Km =5.46±0.41 mM, Vmax=6.16±0.63 mM/h; while the enzyme hydrolyzed Rdâ†'F2, Km=0.603±0.04 mM,Vmax=1.19±0.11 mM/h; hydrolyzed F2â†'C-K, Km=1.25±0.11 mM, Vmax=3.02±0.30 mM/h; hydrolyzed 20(S)-Rg3â†'20(S)-Rh2, Km=0.358±0.019 mM,Vmax=0.056±0.0043 mM/h.The hydrolyzation velocity of multi-glycosly of PPD-type ginsenosides Rbl, Rb2, Rb3, Rc, Rd, F2,20(S)-Rg3 in the same substrate concentration (10 mM) by G-I-g.848 were as follows:while the enzyme hydrolyzes 20-O-Xy1 of Rb3â†'Rd, V0= 0.644 mM/h; while the enzyme hydrolyzes 3-O-Glc of Rb3â†'C-Mxl, V0=4.307 mM/h; the hydrolyzation V0 of Rb1, Rb2, Rc, Rd, F2 and 20(S)-Rg3 was 29.9 mM/h,15.0 mM/h,3.98 mM/h,1.12 mM/h,2.68 mM/h, 0.054 mM/h, respectively. The enzyme hydrolysis velocity (V0) values decrease in the following order:the 20-O-Glc of Rb1> the 3-O-Glc of Rb2> the 3-O-Glc of Rb3 (Rb3â†'C-Mxl path way)> the 3-O-Glc of Rc> the 3-O-Glc of F2> the 3-O-Glc of Rd>the 20-O-Xyl of Rb3 (Rb3â†'Rd path way)> the 3-O-Glc of 20(S)-Rg3.The ginsenosidase G-I-g.48 simultaneously hydrolyzed 3-O-position and 20-O- position glycoside of ginsenoside Rbl or Rb3 with two path way:For Rb1, one path way, hydrolyzed 20-O-Glc of Rb1â†'Rdâ†'F2â†'C-K; another path way, hydrolyzed 3-O-Glc of Rb1â†'Gyp17â†' Gyp75â†'C-K; For Rb3, one path way, hydrolyzed 3-O-Glc of Rb3â†'C-Mx1â†'C-Mxâ†'C-K; another path way, hydrolyzed 20-O-Xyl of Rb3â†'Rdâ†'F2â†'C-K. However, the enzyme hydrolyzed ginsenoside Rb2, Rc, Rd, F2 and 20(,S)-Rg3 with only one path way:firstly, the enzyme hydrolyzed 3-O-Glc of Rb2 or Rc, then hydrolyzed 20-O-Ara, the paty way were Rb2â†'C-Oâ†'C-Yâ†'C-K, Rcâ†'C-Mc1â†'C-Mcâ†'C-K, respectively. The enzyme from A. niger g.48 strain hydrolyzed 3-O-Glc of Rd, F2,20(S)Rg3 path way were Rdâ†'F2â†'C-K, F2â†'C-K, 20(S)-Rg3â†'-20(S)-Rh2â†'Ppdiol, respectively. The kinetic parameters of ginsenosidase type I from A. niger g.48 strain were showed as follow:while the enzyme hydrolyzes 3-O-Glc of Rb1â†'Gyp17, Km=14.6±0.4 mM, Vmax=19.1±1.8 mM/h; while the enzyme hydrolyzes 20-O-Glc of Rb1â†'Rd, Km=15.4±0.3 mM, Vmax=65.4±6.2 mM/h; while the enzyme hydrolyzes 3-O-Glc of Rb2â†'C-O, Km=6.53±0.6 mM, Vmax=24.5±2.5 mM/h; while the enzyme hydrolyzes 3-O-Glc of Rb3â†'C-Mxl, the Km=24.9±2.1 mM, Vmax=33.6±2.8 mM/h; while the enzyme hydrolyzes 20-O-Xyl of Rb3â†'Rd, the Km= 8.78±1.0 mM, Vmax=57.4±5.4 mM/h; while the enzyme hydrolyzes 3-O-Glc of Rcâ†'C-Mcl, Km=4.76±0.38 mM, Vmax=23.3±2.12 mM/h; while the enzyme hydrolyzed Rdâ†'F2, Km= 0.79±0.06 mM, Vmax=11.84±1.3 mM/h; hydrolyzed F2â†'C-K, Km=1.13±0.12 mM, Vmax=3.75±0.36 mM/h; hydrolyzed 20(S)-Rg3â†'20(S)-Rh2, Km=0.16±0.009 mM,Vmax=0.22±0.015 mM/h.The hydroxylation velocity of multi-glycosly of PPD-type ginsenosides Rbl, Rb2, Rb3, Rc, Rd, F2,20(S)-Rg3 in the same substrate concentration (10 mM) by G-I-g.48 were as follows: while the enzyme hydrolyzes 20-O-Xyl of Rb3â†'Rd, V0=30.6 mM/h; while the enzyme hydrolyzes 3-O-Glc of Rb3â†'C-Mx1, V0= 9.64 mM/h; while the enzyme hydrolyzes 20-O-Glc of Rb1â†'Rd, Vo= 25.7 mM/h; while the enzyme hydrolyzes 3-O-Glc of Rb1â†'Gyp17, V0=7.75 mM/h; the hydrolyzation V0 of Rb2, Rc, Rd, F2 and 20(S)-Rg3 was 14.8 mM/h,15.9 mM/h,11.0 mM/h,3.37 mM/h,0.22 mM/h, respectively. The enzyme hydrolysis velocity (V0) values decrease in the following order:the hydrolyzation velocity of 20-O-Xyl of Rb3 (Rb3â†'Rd path way)>20-O-Glc of Rb1(Rb1â†'Rd path way)>3-O-Glc of Rc>3-O-Glc of Rb2> 3-O-Glc of Rd>3-O-Glc of Rb3 (Rb3â†'C-Mxl path way)> 3-O-Glc of Rb1(Rb1â†'Gyp17 path way)> 3-O-Glc of F2>3-O-Glc of 20(S)-Rg3.Both ginsenosidase G-I-g.848 and G-I-g.48 can hydrolyze the 3-O- and 20-O-glycosides of PPD type ginsenosides, however, their hydrolyzation pathway exhibit some different:when hydrolyzing Rb1, G-I-g.848 firstly hydrolyze the 20-O-Glc of Rb1, then hydrolyze the 3-O-Glc with only one pathway; G-I-g.48 can hydrolyze the 3-O- and 20-O-Glc of Rbl at the same time with two path way. The hydrolyzation velocity of 3-O-glycosides of PPD type ginsenosides by G-I-g.848 were faster than that of 20-O-glycosides (except Rbl); but the hydrolyzation velocity of 20-O-glycosides of PPD type ginsenosides by G-I-g.48 were faster than that of 3-O-glycosidesThe kinetic study of these two enzyme suggested that we can obtain the desired specific ginsenoside such as ginsenoside F2, C-Y, C-Mc, C-Mx, C-K by regulating the reaction time and the substrate concentration, from ginsenosides Rbl, Rb2, Rb3, Rc, Rd. In this thesis, low-cost PPD type ginsenoside of American ginseng (mainly contain Rbl, Rb2, Rc and Rd) was hydrolyzed by low-cost crude enzyme from A. niger g.848 strain to obtain high-active rare ginsenoside C-K, F2, C-Y and C-Mc; Ginsenosides of Notoginseng leaf (mainly contain Rb3, C-Mx1) was hydrolyzed by low-cost crude enzyme from A. niger g.848 strain to obtain high-active rare ginsenoside C-K and C-Mx. The optimal enzyme reaction conditions: substrate concentration was set at 3%, reaction temperature was 45℃, pH 5.0, and reaction for 18 h. Finally,50 g of rare ginsenoside mixture was obtained from 80 g PPD type ginsenosides of American ginseng; then,5.20 g of monomer ginsenoside C-Mc,0.96 g of C-Y,16.3 g of F2 and 16.9 g of C-K were isolated and purified from the mixture by silica gel column separation; the purity of above monomer ginsenosides were more than 90% in HPLC. The mole yield of C-Mc from Rc was 43.7%; C-Y from Rb2 was 42.4%; F2 and C-K from Rb1 and Rd was 69.5%.31.0 g of rare ginsenoside mixture was obtained from 50 g of ginsenosides of Notoginseng leaf; then,9.43 g of monomer ginsenoside C-Mx,5.45 g of C-K were isolated and purified from the mixture by silica gel column separation; the purity of above monomer ginsenosides were more than 90% in HPLC. The mole yield of C-Mx from Rb3 and C-Mxl was 48.2%; C-K from other ginsenosides was 41.2%. The above preparation of monomer ginsenosides were very successful, and provide the basis for industrialization.Part II The biotransformation of high-content Salvianolic acid in danshenIn order to obtain Przewalskinic acid A and danshensu (mainly Danshen hydrosoluble composition with high-active and small molecule), in this part, the Salvianolic acid-esterase production by microbial fermentation, enzyme separation and purification, enzymes Catalytic properties were studied; and the preparation of Przewalskinic acid A and danshensu from Salvianolic acid B by the Salvianolic acid-esterase from Aspergillus oryzae D30s strain was studied.Aspergillus oryzae D30s, used as the enzyme producing microorganism, was fermented; after centrifugation, impurities were removed from Fermented liquid, then the crude enzyme was precipitated by the method of ammonium sulphate precipitation. The optimal fermentation conditions were confirmed as follows:the medium contains 5% of wort; fermentation temperature was 40℃; fermentation time was 96 h using shake cultivation. After removing cells and concentration, two kind of Salvianolic acid-esterase were purifited from the fermentation liquor by the method of DEAE-cellulose-DE52 anion exchange resin and active-PAGE. The molecular weight of Salvianolic acid-esterase El and E2 was 36 kDa and 65 kDa based on SDS-PAGE, respectively. Both the Salvianolic acid-esterase El and E2can hydrolyze Salvianolic acid B to danshensu and Przewalskinic acid A. In this thesis, Salvianolic acid-esterase E1(M.W.,36 kDa) was mainly studied:the optimum pH was 5.0, and the enzyme was stabilized in pH 4.0-7.0; the optimum temperature was 40℃, and the enzyme was stabilized under 50℃. This enzyme shows a special hydrolytic activity on the ester bond of Salvianolic acid B, but can not hydrolyze pNPP. The Salvianolic acid-esterase from Aspergillus oryzae D30s strain was different from other reported esterase, and also has not been reported in NC-IUBMB (http://www.chem. qmul.ac.uk/iubmb/), so, the enzyme was a new kind of esterase.Finally, the study of biotransformation of Salvianolic acid B to high activity of Przewalskinic acid A and danshensu by the crude Salvianolic acid-esterase from Aspergillus oryzae D30s strain was investigated. The enzyme solution was reacted with 1.6% of substrate for 14 h at pH 5.0 and 40℃ After reaction, the enzyme was retrived to next reaction, and the supernatant was purified by Sepabeads SP207 macroporous resin to obtain the monomer of enzymatic hydrolysis products:Przewalskinic acid A and danshensu.31.3 g of Przewalskinic acid A (91.0% purity) and 13.1 g danshensu (95.0% purity) were obtained from 75 g of salvianolic acid B. The preparation of Przewalskinic acid A was very successful which yield was over 86% of theoretical yield, but the yield of danshensu was only 33%. UPLC-MS and NMR mothed were used to the future check with the structure of danshensu and Przewalskinic acid A.In this thesis we studied two kinds of novel ginsenosidase type â… , namely G-I-g.848 and G-I-g.48, from Aspergillus niger g.848 and Aspergillus niger g.48 and a new Salvianolic acid-esterase from Aspergillus oryzae D30s strain. We built up the theoretical system of enzyme catalysis of these enzymes. Using the crude ginsenoside type I, the rare ginsenosides C-K, F2, C-Y and C-Mc with high activities such as anticancer, antithrombotic and resistance to diabetes were prepared. And, the high activity Przewalskinic acid A and danshensu were also prepared. This thesis provided an important theoretical evidence and useful method for developing a series of ginseng product, drugs, health food, and cosmetics.