Microbial Transformation Of Astragalosides To Astragaloside â…£by Absidia Corymbifera AS2

Astragaloside IV (ASI) is believed to be the main active constituent that is normally used as a marker for quality control of Radix Astragali. ASI has been proved to have special biological activities. A new medicine, ASI glucose injection, for treatment of ischemic myocarditis is on the third period clinical research. Now ASI is mainly obtained from plant extraction for lacking of total synthesis method. Due to the low content of ASI in natural plants, the isolation of ASI from natural products is extremely difficult and cost.Microbial transformation which has high specificity and environmental compatibility is increasingly being used as a useful tool in the structure modification of natural compounds. In this study, a new method by microbial transformation to enrich ASI was reported. A new deacetylase was purified from A. corymbifera AS2by combination of different traditional protein purification chromatographics. Four precursors were identified and a new hydrolyzing pathway of these four precursors by the purified deacetylase was conjectured. The dissertation is thankful to the financial support of Chinese National Science and Technology Major Project (2009ZX09301-011).1, Absidia corymbifera AS2, which has the highest ability of biotransforming astragalosides to ASI was screened from various fungi. Total astragalosides as substrate with the concentration of5g/L, transformed for60h, the mol degree of conversion arrived at90.3%in5L flask. The yield of ASI was improved by about3folds and the product of ASI was mainly found in the supernatant. A simple, stable and effective method for isolation and purification of ASI from fermentation medium with D101resin was established. After purification, the chromatographic purity and recovery of ASI arrived at95%and62%, respectively.2.. The precursors of astragalosides which can be transformed to ASI by microorganisms were investigated. By the methods of silica gel and reverse-phase silica gel chromatography and microbial transformation tracking, Astragaloside I (AS-Ⅰ), Isoastragaloside Ⅰ (isoAS-I), Astragalside Ⅱ (AS-Ⅱ) and Isoastragaloside Ⅱ (isoAS-Ⅱ) were finally separated and identified. These four precursors were analyzed by HPLC-ELSD-DAD to assure the quantity level. A convenient HPLC method for studying the metabolic pathways was established and used to study the metabolic pathways of ASI, AS-Ⅰ, isoAS-Ⅰ, AS-Ⅱ and isoAS-Ⅱ.3、Compared with the chemical structure of ASI, there are two more acetyls on3-xylose residue of AS-Ⅰ (2’3’-di-OAc) and isoAS-Ⅰ (2’4’-di-OAc), and one more acetyls on3-xylose residue of isoAS-II (3’-OAc) and AS-Ⅱ (2’-OAc). It points that A. corymbifera AS2has deacetylases which can hydrolyze acetyls residues of some astragalosides to ASI. The purification of deacetylases from A. corymbifera AS2was continued to be studied. Finally, one deacetylase was purified from A. corymbifera AS2by combination of different traditional protein purification chromatographics: ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic chromatography, CHT ceramic hydroxyapatite and Resource Q prepacked column. The activity recovery and the protein recovery of the purified enzyme were0.32%and0.075%o, respectively.4、The properties of the purified enzyme were also studied. The molecular weight (MW) of enzyme subunits was38KDa by SDS-PAGE and36067Da by MS. The purified deacetylase had optimal activities at pH8.0and was stable within pH7.0-9.5. The enzyme had optimal temperature of35-45and was stable lower than45℃. The kinetic parameters, Km and Fmax values, of the purified enzyme against p-NPA were determined by typical Lineweaver-Burk double reciprocal plots under pH7.0and30℃. The Km values was3.76mmol/L and the Vmax values was17.64mmol/min/mg. The amino acid sequences of deacetylase were further analyzed with the PSD-MALDIMS method sulfonated by SPITC.5、The hydrolyzing pathway of the four precursors by the purified deacetylase was further studied. The result showed that the hydrolyzing pathways were AS-Ⅱ→ASI; isoAS-Ⅱ→-AS-Ⅱ→ASI; AS-Ⅰ→(AS-Ⅱ、isoAS-Ⅱ)-ASI; isoAS-Ⅰ-AS-Ⅱ-ASI。...