Enzymatic Production Of Glycyrrhetinic Acid And The Separation Process Design

Glycyrrhetinic acid(GA)is a derivative generated by hydrolyzing two glucuronide groups from glycyrrhizic acid(GL).GA has anti-inflammatory,enhancing immunity,antiarrhythmia,anti-cancer,anti-tumor,anti-virus and other functions,so it has been widely used in food,medicine,cosmetics and other industries.Currently,GA is mainly obtained by the direct extraction from the licorice or the acidic/alkaline hydrolysis of GL or chemical synthesis.Among them,the direct extraction from licorice is simple,but the GA content is very low with mass fraction of only(0.01% to 0.2%)which results in the low purity and yield of the product and also causes extra difficult to the downstream separation.The acid hydrolysis of GL gives high GA purity,but it requires harsh reaction conditions such as high temperature and high pressure.Recently,enzymatic preparation of GA has received special attention due to the gentlereaction conditions,good chemical bond selectivity and high specificity.The main purpose of this thesis is to prepare GA by enzymatic hydrolysis of GL catalyzed by ?-glucuronidase from Aspergillus oryzae(PGUS-E)and design separation process for GA.The main results are as follows:First,we investigated the thermostability,optimal reaction temperature and optimal pH of PGUS-E,and the results showed that the optimal reaction temperature of PGUS-E was 40 °C,and optimal pH of PGUS-E was 5.0.On this basis,we explored the preparation method of GA by using PGUS-E catalysis.Through the optimization of different conditions,we obtained the optimum preparation conditions of GA : the optimal ratio of enzyme and substrate(1:3),rotational speed(250 rpm),pH(5.0),reaction time(6 h),temperature(40 °C).Under these optimum conditions,the conversion ratio of GL and yield of GA reached 96.4% and 93.8%,respectively.This process has several advantages including short operation time,high conversion ratio and yield,and good reproducibility.Then,we carried out the reaction at 30 L scale,and the results were consistent with that of small scale reaction,which provided the basis for industrialized production.Finally,we developed a protocol to separate GA from the reactants.Under optimal conditions(pH 5.0,85% ethanol),the GA purity of 93.6% was obtained.Furthermore,through ethanol recrystallization at low temperature,the GA purity reached as high as 96.9%.Therefore,the separation process proposed in this study has several advantages including low cost,high yield and less pollution,which can solve the current problems in GA production such as uncontrollable acidic hydrolysis condition,long processing line,low purity of product and serious environmental pollution.