Biotransformation And Purification Of Glycyrrhetinic Acid Monoglucuronide

In order to produce Glycyrrhetinic Acid Monoglucuronide (GAMG) by Microbialtransformation from Glycyrrhizin (GL), four kinds of biological mold were isolated fromlicorices. By using preliminary screening culture medium, four microbe were targetmolds.Then by using the rescreening culture medium, one high-efficiency transformationmold for transformating GL into GAMG was determined from the four kinds of biologicalmolds, and they were identificated to be the Aspergillus.sp. by the determination of18SrDNA sequencing.In the paper, HPLC method was used to analysis GL and GAMG under theconditions: Agilent-ExtendC18(4.6mm×250mm,15μm) served as the stationary phase;acetonitrile-water served as (80:20, v/v) the mobile phase; The UV detection wavelengthwas254nm; the flow rate was1.00mL/min; the column temperature was25℃. Thestandard curve of GL and GAMG was y=0.2308x-6.2082, R2=0.9987andy=0.1694x+20.421, R2=0.99943, respectively.Based on the research of Aspergillus.sp IS254, PB experiment, Steepest ascent designand Response Surface Method were selected to analysis the optimum factor level,decidedthe obvious influce facor and optimize the fermentation conditions, respectively. Theoptimum fermentation conditions was as following: GL was1.19g, NaNO3was2g,MgSO4was0.7g, K2HPO4was1g, FeSO4was0.01g in the1L fermentation liquid with pHwas5.5, the inoculum amount of the high-efficiency transformation mold is10mL,temperature was28℃after6days the transformation rate could reach to35.72%.The β-Glucosidase was extracted within the fermentation liquid, and the enzymologyproperties of which was studied. The results showed that when the optimal temperaturefor the enzymatic hydrolysis was50℃, the optimum pH was5.5, the conversion rate was42%. The β-Glucosidase was stable, when the temperature was between4℃-50℃, andthe pH ranged from5to8.Product transformated from the high-efficiency transformation mold was separatedand purified by silica gel column chromatography and macroporous resin HP20colummseparation technology. The optimum condition was ascertained. For silica gel columnchromatography, chloroform: methanol: acetic acid glacial=70:30:1(v/v/v) was the eluent,the flow rate was2mL/min, the sample amount was1.5mL, the purity of GAMG was47.3%. When by using the HP20macroporous resin columm, different concentrations ofethanol served as the eluent, under the conditions of the high-diamter8.5, the flow rate1.5mL/min, the sample amount2mL, the purity of GAMG was57%.