Preparation Of Cycloastragenol By Biological Method

The "telomere theory" has a great influence on the study of aging,and the role of telomerase in lengthening telomeres has been recognized.Cycloastragenol is the aglycone of Astragaloside IV and most astragaloside saponins.It is the only compound that can activate telomerase and has anti-aging and immune-enhancing effects.Therefore,it has a broad application prospect in the medical field.Considering that the main preparation methods of Cycloastragenol——acid hydrolysis,Smith degradation and enzymatic hydrolysis——have the disadvantages of high cost and high pollution,and there are few methods for microbial preparation of Cycloastragenol,so the paper uses microbial method,and successfully screened a strain capable of efficiently hydrolyzing Astragaloside IV to prepare Cycloastragenol.Cycloastragenol is obtained by hydrolysis of Astragaloside IV to break the β-glycosidic bond between the C3 xylose and C6 glucose.According to the weak specificity of β-glucosidase substrate,this paper first screened out the strains that could express glucosidase with high enzyme activity,then cultured Astragaloside IV as the substrate,screened out the strains that could efficiently hydrolyze Astragaloside IV to Cycloastragenol,and optimized the hydrolysis and transformation conditions.This content of the paper is divided into the following two parts.(1)This paper first tried to screen the strain from the soil.A screening method was established to screen the strains with higher activity ofβ-glucosidase as preliminary screening,and then to screen the strains hydrolyzed Astragaloside IV to obtain Cycloastragenol as secondary screening.But after constant trials,the ideal strain was not obtained.Secondly,four strains that could hydrolyze Astragaloside IV were successfully screened from the decayed Astragalus by using the screening method.The four strains were identified as Fusarium proliferatum,Fusariumi verticillioides,Talaromyces funiculosus and Talaromyces stollii.The Fusarium proliferatum and Fusarium verticillioides can hydrolyze the glucose on the Astragaloside IV molecule and obtain the intermediates 3-O-xy lo-Cycloastragenol.The Talaromyces funiculosus and Talaromyces stollii can hydrolyze xylose from Astragaloside IV molecule to get 6-O-glucose-Cycloastragenol.Based on this result,it is speculated that the reason for not obtaining Cycloastragenol is that the amount of enzyme secreted by the cells is not sufficient.Therefore,the seed liquids of the four strains and the transformation medium having a volume ratio of 100%were filtered,and then the cells were added to the transformation medium for transformation culture,and the same operation was carried out on the pairwise combination of the four strains in the ratio of 1:1,and the strain Talaromyces stollii is finally screened,and the yield of Cycloastragenol is 0.73 mg,the conversion rate of Astragaloside IV is 23.36%.(2)The transformation medium composition and transformation conditions of the selected strain Talaromyces stollii hydrolyzed Astragaloside IV to prepare Cycloastragenol were optimized,and finally the optimal transformation medium was:Astragalus powder 0.80%,peptone 0.80%,KH0PO4 0.04%,FeSO4 0.04%,Astragalus membranaceus extract 0.02%,after sterilization,0.20%methanol was added during inoculation;the optimal transformation conditions were:the seed liquid is added to the transformation medium after filtering with a seed liquid having a volume ratio of 100%to the transformation medium,initial pH of the medium 5.0,liquid volume 50 mL/250 mL,culture temperature The culture speed was 150 r/min at 35℃,and the culture time was 5 days.Under the optimal conditions,the yield of Cycloastragenol reached 2.738 mg,and the conversion rate of Astragaloside IV reached 87.589%,which was 3.75 times higher than that before optimization.