The Conversion Of Stevioside By Two Fungi And The Separation And Purification Of Related Enzymes

Stevioside is a kind of natural terpenoids indicant sweeteners extracted from stevia rebaudiana bertoni leaf.The steviol,aglycone of stevioside,has antipodalene kauri tetracyclic diterpene skeleton structure.Steviol is one of the molecular skeleton of new drug research.This work make the transformation of stevioside by Aspergillus aculeatus and Penicillium poinophilum and the purification of the related invertase.The Aspergillus aculeatus enzyme extracts could convert the stevioside and rebaudioside C to a new compound within 10 hours.The conversion rate was 98.0%in 24 hours.The conversion product existed in deposit was identified as steviol.The purity and recovery percent of steviol were 95.2%and 84.0%respectively.Steviol could occur to deposit,Raudioside A existed in supernatant could be purified easily.RA could be obtained by resin adsorption separation purification and the recovery rate and the purity could reach 80.5%and 86.2%respectively.This work research the conversion of stevioside by Aspergillus aculeatus.The mycelium of Aspergillus aculeatus can convert the rebaudiana C with in four days specifically and the conversion rate is 97.9%with the new compound Rubusoside.Extending the time to the conversion rate of stevioside was 5.0%but the RA remained stable.The conversion rate of stevioside in supernatant liquid which removed hyphae from zymotic fluid was only 2.1%.The conclusion of that was the invertase of RC mainly existed in hyphae.Aspergillus aculeatus cultivated by inducing medium,centrifuging,removing hyphae and getting the supernatant.The adding 1.0%stevioside to the supernatant and the research findings show that no conversion effect.Above all,the experimental results indicated that the different enzymes with different culture conditions.The enzyme extracted from Aspergillus aculeatus by solid cuture was purified by a series of treatments such as dialysis,anion-exchange chromatography and molecular sieve and gel filtration chromatography purification,etc.Under 50%-60%ammonium sulfate saturation,enzyme has strong ability of transformation of stevia sugar.The protein molecular weights between 30kd-60kd by protein gel electrophoresis analysis.Specific activity of enzymes gradually rise and the purity increased by 20.6 times and the energy increase to 4.8.The penicillium pinophilum enzyme extracts could convert the stevioside firstly to a new product within 18h and the conversion rate was 100%.Then the enzyme could convert the RC to the same product with in 50h and the conversion rate was 100%.RA remained unchanged.The penicillium pinophilum hyphae could covert 1%stevioside,which contains 50%SS,12.5%RC,37.5%RA under constant temperature 30 ?.The converted compound was rubusoside,the SS,RC and RA conversion rate were 41.8%,4.9%and 4.7%respectively.A series of stevia rebaudianas were analysis by HPLC method.The results show N0.7 that the total glycoside reached 14.2%,RA 8.2%,SS 5.1%,RC 0.8%.The total stevia of No.7 sample was higher than related content from ZS-1(glycosides 12.39%,RA 7.69%).The variety of research significance is one of the worthy varieties.