Biosynthesis Of Glycyrrhetic Acid 3-O-mono-?-D-glucuronide By The Endophytic Fungi From Dong-xiang Wild Rice

Glycyrrhizin?GL?,the principal active ingredient of licorice,has many bioactivities,such as anti-bacterial,anti-inflammatory,anti-viral,anti-tumor and so on.Glycyrrhetic acid 3-O-mono-?-D-glucuronide?GAMG?,the derivative of GL,exhibits stronger physiological properties?anti-cancer,anti-anaphylactic,anti-viral and so on?and a wider spectrum of biological activities as compared to GL.Furthermore,GAMG is a new type sweetener with high intensity sweetness and low calorie,and safer than GL.Therefore,GAMG possess a more important research value and a better prospect of application especially in pharmaceutical industry and food industry.With an abundant biology diversity,the endophytic fungi is an untapped biological catalyst treasure.the researches on biotransformation of GL to GAMG by the endophytic fungi from plants were rarely been reported so far.In this study,17endophytic fungi from Dong-xiang wild rice were tested for their capablities of transforming GL to GAMG in a medium containing GL as the sole carbon source.And the strain S108 with the strongest ability was selected to furtherly study,including follows contents:1.A ultra performance liquid chromatography?UPLC?method was developed to simultaneously determined the contents of GL and GAMG in broth.The optimal chromatographic conditions was as follows:Waters Acquity system?Waters,USA?with an auto injector,an C₁₈,5?m,4.6 mm×250 mm chromatographic column,the wavelength was set at 254 nm,the mobile phases was 80%methanol containing 0.5%acetic acid?v/v?,the injection volume was 10?l,the flow rate was 1 mL/min,the column temperature was 35?C.The results showed that when the sample amount range was between 0.2?g and 20?g,the detection precision of GL and GAMG was high.2.Regarding 17 strains endophytic fungi from Dong-xiang wild rice as the object of study,screening strains for transforming GL in a medium containing GL as the sole carbon source was carried out.The result showed that 9 strains were able to transforming GL,the three of them could transform GL into GAMG,and the strain S108 with the strongest ability in biosynthesis of GAMG.Combined morphology with ITS-r DNA systematical analysis,the strain S108 was identified as Chaetomium globosum S108.3.Optimization of production of?-D-glucosidase by Chaetomium globosum S108.The result showed that the?-D-glucosidase was induced and synthesized by glycyrrhizin and was an extracellular enzyme.The optimal condition for producing?-D-glucosidase was as follows:glycyrrhizin 2 mg/mL,organic nitrogen source was peptone,inorganic nitrogen source was NH₄NO₃,initial pH 5.0,temperature 32?C,rotate speed 140 rpm and inoculum volume 2%.4.The isolation of the crude?-D-glucosidase from S108 and the study on the enzymology characteristics of it was carried out.The results showed that the optimal reaction temperature was 50?C,the optimum pH was 5.0.When the temperature was between 25⁵0?C,and the pH ranged from 4.0 to 8.0,the?-D-glucosidase was stable.Zn²⁺and H₃BO₃ were activators,while Fe²⁺and Cu²⁺were inhibitors of purified?-D-glucosidase.5.Optimization of biotransformation condition for the maximum molar yield of GAMG.Single factor experiment design and PB experiment were used to find out the significant factors.And steepest ascent design was selected to confirmed the central point.Finally,BB experiment and response surface method were used to give the optimal biotransformation condition.The result showed that the optimal biotransformation was as follows:glycyrrhizin was 1.96 g/L,initial pH 6.0,inoculum volume 3.96%.Thus a yield of 98.68%for GAMG was obtained which 22.16%higher than initial biotransformation.