Biotransformation Glycyrrhizic Acid To Monoglucuronyl-glycyrrhetinic Acid By Mutation Of Aspergillus

Glycyrrhizic acid(GL) is one of the major components of liquorice root(Glycyrrhice glabra) which has certain physiological activity. Biotransformation of Glycyrrhizic acid to glycyrrhetic acid Glycyrrhetinic acid monoglucuronide(GAMG) is one of the effective ways to obtain higher physiological activity components like the latter.By UV and UV-nitrous acid combined mutation, a strain of Aspergillus Niger was screened. Conversion rate from GL to GAMG was 54.14% and 14.1 times higher than the original starting strain. Through plotting fatality rate curve, optimum conditions for mutation of Aspergillus niger IS254 were as followed: irradiated from2.5 min to 4.5 min, and treated with 200 mmol/L HNO2 for 40 min.Through ultraviolet mutation of Aspergillus oryzae and plot the fatality rate curve, optimum conditions were determined: the best mutation time is for 4 min. Screened strains to convert GL to GAMG efficiently with the conversion rate 25% and11.4 times than that of starting strain. By determination 18 S rDNA, the similarity of Aspergillus oryzae IS729 with Aspergillus oryzae SEMCC-3.248 is 99.0 %. The optimum fermentation conditions was performed by single factor experiment and got the results as followed: time of incubation was 5 days, NaNO3 was 2.5 g/L, MgSO4 was 0.7g/L, K2HPO4 was 1 g/L, GL 1.2 g/L, temperature was 30 ℃, fermentation liquid pH was 5.5, quantity of vaccination 10 ml, peptone was 1.5 g/L and FeSO4 was 0.01 g/L.β-glycosidase enzymes produced by Aspergillus Niger was purified by the method of ammonium sulfate fractional precipitation and dialysis filtering and to explore its enzymatic properties. Enzymatic analysis results showed that the optimum reaction temperature for β-glycosidase enzymes produced by Aspergillus Niger was to be 60 ℃,and then was the excellent thermal stability.within the range of 40 ~ 60℃.The most optimum pH for β-glycosidase enzymes was 6.0, while it was stability under the acidic conditions in which pH range from 4.0 to 7.0. Conversion rate of GL to GAMG was stability. GL as the substrate, study the enzymatic reaction kinetics, Vmax = 0.26 g/L/d, Km = 0.83 g/L.In order to investigate the process of bioconvertion GL to GAMG, it is necessary to establish a method for detecting components of GL fermented liquid. The experiment use Extend-C18(4.6 mm×150 mm, 5 μm) as stationary phase; mobile phase, methanol: 3.60 % acetic acid aqueous solution 85:15(v/v); column temperature, 25 ℃; the flow rate,1.00 ml/min; detection wavelength, 254 nm, injection amount 5 μL. Under the chromatographic conditions, the sample is separated very well. Glycyrrhizic acid and glycyrrhetinic acid in a range of 0.2 ~ 182.4 mg/L and 0.3 ~ 118.8mg/L present a good linearity relationship, respectively. Glycyrrhizic acid and glycyrrhetinic acid with respect to the average recovery was 104.22% and 105.58%, respectively.The analysis method is simple, sensitive and accurate for investigating the biotransformation of GL fermentation process and production quality control of the GAMG.