Ide Rb1, Rb2, Rc, and Rd. Rb1, Rb2, Rc, Rd, F
Ide Rb1, Rb2, Rc, and Rd. Rb1, Rb2, Rc, Rd, F2, C-K, C-Y, and C-O, C-Mc, and C-Mc1, standard ginsenosides. 1e3, enzyme reaction product from 25mM Rb1, 25mM Rb2, 25mM Rc reacted at 45 C for 3 h; 4, enzyme reaction solution from 2.5mM Rd. Solvent, chloroform:methanol:water sirtuininhibitor7.five:2.five:0.5; 10 H2SO4 as a chromogenic agent.shown). Inside the purification, the yield of your ginsenosidase was about three.1 , and also the particular activity from the enzyme increased 13 times (data not shown). three.2. Pure enzyme hydrolysis on the monomer ginsenosides Rb1, Rb2, Rc, and Rd The enzyme from the A. niger g.848 strain reacted with 25mM monomer ginsenoside Rb1, Rb2 and Rc at 45 C for three h, respectively; and reacted with 2.5mM Rd at 45 C for 0.5 h. The enzyme reaction goods were examined by TLC (Fig. two). As shown in Fig. 2, the ginsenosidase made by A. niger g.848 strain firstly hydrolyzed the 20-O-b-D-(1/6)-glucopyranoside of Rb1 into Rd, then hydrolyzed the 3-O-b-D-(1/2)-glucopyranoside of Rd to F2, HSPA5/GRP-78, Human (His) further to C-K. Nonetheless, the enzyme firstly hydrolyzed the 3-O-b-D-(1/2)-glucopyranoside of Rb2 into C-O, hydrolyzed 3-O-b-D-glucopyranoside of C-O into C-Y, further hydrolyzed theAB0.40 0.30 AU 0.20 0.ten 0.00 2.00 4.6.six.8.00 Minutes10.12.14.Fig. 1. Purified ginsenosidase Adiponectin/Acrp30, Mouse (227a.a) type-I in SDS-PAGE and HPLC. (A) Ginsenosidase Type-I SDS-PAGE; marker, marker protein: phosphorylase b (97.2 kDa), serum albumin (66.4 kDa), ovalbumin (44.three kDa), carbonic anhydrase (29.0 kDa), trypsin inhibitor (20.1 kDa), and lysozyme (14.3 kDa). Protein quantity, three mg. (B) Ginsenosidase Type-I HPLC. HPLC, higher performance liquid chromatography; Web page, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate.C.-Y. Liu et al / Minor ginsenoside preparation20-O-a-L-(1/6)-arabinopyranoside (arap) of C-Y into C-K; the enzyme also firstly hydrolyzed the 3-O-b-D-(1/2)-glucopyranoside of Rc to C-Mc1, hydrolyzed the 3-O-b-D-glucopyranoside of CMc1 into C-Mc, and further hydrolyzed 20-O-a-L-(1/6)-arabinofuranoside (araf) of C-Mc into C-K. The biotransformation pathway of your ginsenosidase produced by A. niger g.848 strain is shown in Fig. three. Consequently, the enzyme from A. niger g.848 strain can hydrolyze the 3-C position (3-O-) and 20-C-position (20-O-) multiglycoside of PPD-type ginsenosides like Rb1, Rb2, Rc, and Rd, and should be classified to ginsenosidase type-I created by Aspergillus sp.48 strain [23] as well as a. niger g.48 strain [24]. Having said that, the hydrolysis pathway from the specific ginsenosidase type-I from A. niger g.848 strain in present study is distinctive with that of ginsenosidase type-I from A. niger g.48 strain; the ginsenosidase type-I (molecular weight, 75 kDa) hydrolysis pathway from A. niger g.848 strain on ginsenoside Rb1 (within this study) is Rb1/Rd/F2/C-K; but the ginsenosidase type-I (molecular weight, 74 kDa) from A. niger g.48 strain hydrolyzes both 3-O- and 20-O-glucosides of Rb1 with two pathways: i.e., 1 pathway was Rb1/Rd/F2/C-K; a different, Rb1/Gyp17/Gyp75/C-K [24]. As a result, the enzyme A. niger g.848 strain is usually a special ginsenosidase type-I differentiating with the ginsenosidase type-I from A. niger g.48 strain. The ginsenosidase type-I from A. niger g.848 strain is are appropriate than that of A. niger g.48 strain, for the reason that the ginsenoside Rb1 is hydrolyzed by ginsenosidase type-I from A. niger g.848 strain with 1 pathway, along with the enzyme from A. niger g.48 strain hydrolyzes Rb1 with two pathways [24].3.three. Enzyme reaction kinetics To ascertain the enzyme kinetic parameter.
