Research On Production Of Betulinic Acid Through Biotransformation By Armillaria Luteo-virens Sacc

Betulinic acid has attracted attention recently due to its broad physiological and pharmacological properties, such as anti-HIV and anti-cancer activities. In this thesis, the present research is focused on the biotransformation of betulinic acid from betulin by Armillaria luteo-virens Sacc.In order to obtain suitable biocatalyst for betulinic acid producing, Armillaria luteo-virens Sacc ZJUQH was bred by low energy N+ implantation methods. As a result, a genetically stable mutant strain, designated as ZJUQH100-6, was obtained with the productivity of betulinic acid reaching 5.34%. Micromorphology was significantly different between the mutant and the original one by scanning electron microscopy (SEM).In the optimization procedure for conversion conditions, seven kinds of conditions were designed. Results showed that the transformation caused by pre-cultured fungal mycelia, designated as G2, was favorable for betulin conversion as the productivity of betulinic acid was evaluated. Subsequently fractional factorial design (FFD) and response surface methodology (RSM) were applied to optimize the main parameters that affect betulinic acid production in the growing-cells system. The central composite experimental design was then adopted to derive a statistical model for optimizing biotransformation conditions. The optimum conditions were observed at pH 5.0,0.57% Tween 80,15 mg/L betulin and 3 days of stage of inoculation. Under the optimized conditions, the highest productivity of betulinic acid predicted was 9.32%.Moreover, an efficient ionic liquid-containing system (ILs) has been developed for betulinic acid formation catalyzed by resting cells of Armillaria luteo-virens Sacc ZJUQH100-6. The optimum conditions were as follows:5 mL of [EMIM][BF4]/hexane system (v/v=1:1),0.1 M butanol as co-substrates,75 mg/L betulin,300 g/L resting cells and inoculation for 18 h. As compared with aqueous system, the yield of betulinic acid in ILs system was higher. More importantly, the reaction time was less, indicating that ILs are promising candidates for using as the second solvent in betulinic acid production by whole-cells biocatalysts.The Cytochrome P450 enzyme involved into biosynthesis of betulinic acid was also preliminary studied. The solvent n-Hexane was certified to be efficient for P450 inducing. The P450 and P420 concentration in microsomes were highest when 2μL/mL·h of n-Hexane was added for 9 h, which were detected by CO difference spectra method.In addition, in order to obtain qualitative and quantitative analysis of betulin and betulinic acid in biotransformation system, simualtaneously, the ultraviolet spectrophotometry and RP-HPLC analysis methods were set up. They provide the base for other studies.