Penicillium Fungi J - 9 Beta Glycosidase Enzymes Fermentation Research And Their Transformation Products Asiatic Acid Separation And Detection Of Total Single Glycosidase

Penicillium fungi J-9can produce β-glucosidase, hydrolyzebeta-1,6-glycosidic bond of total glucosides of Centella asiatica. Ittransforms total glucosides of Centella asiatica into asiaticacid–28-O-β-D-glucopyranoside,centelloside C and chebuloside II. Thisarticle optimized the liquid fermentation process and the extractionand separation technology of the β-glucosidase.Then stude theseparation and purification,structural identification and quantitativeanalysis of the three transformed products from the total glucosides ofCentella asiatica.First,using the principle of the β-D-glucosidase transforming pNPGinto pNP by hydrolyzing glycosidic bond,and the combination of pNPand Na2CO3can be detected in400nm,we can assay the enzymeactivity of β-D-glucosidase.To optimize the enzyme activity,wecompared the influence of the type and the amount of the carbonsource,the pH values of the medium,the concentration of thesubstrate,temperature, loading amount of medium and the timelengths of fermentation on the production of β-D-glucosidase.Theoptimized the β-D-glucosidase fermentation process is as follows:Theculture medium is consist of3%wheat bran and2%lactose,and theinducer is3mg/mL total glucosides of Centella asiatica,adjust themedium initial pH to5.7,cultural temperature is32℃,200r/min shakingfermentation for7days.In these conditions, the enzyme activity of β-glucosidase produced by J-9strain comes up to1.4U,is5times of theinitial PDA medium.We find the optimized extraction process of the β-glucosidase bycomparing the precipitation methods of ammonium sulfate, ethanoland acetone on the enzymeactivity of β-glucosidase,and the pH ofbuffer solution on DEAE-52sepharose purification performance. Centrifuge the fermentation broth to separate mycelia andsupernatant,add30%saturation of ammonium sulfate to precipitatingenzyme,centrifuge,put the precipitate into the dialysis bag,adjust thepH of the dialysate to7.5by Tris-HCl buffer solution,load it onto DEAE-52Sepharose ion exchange chromatography column,then elute with0.1MNaCl-Tris-HCl buffer solution,concentrate the eluate byultrafiltration,load it into onto SDS-PAGE gel electrophoresis to detectthe purity and molecular weight of β-glucosidase.The result shows thatthe purified β-glucosidase is composed of two bands,and themolecular weight of the main band is65.0kDa.Then we study onphysical and chemical properties of the β-glucosidase,and investigatethe impact of factors such as temperature,pH, metal ions on enzymeactivity.The results show that the optimum pH is4.5,the optimumtemperature is50℃,and the β-glucosidase is stable at20℃.Metal ionshave different effects on the enzyme activity, K+shows the highestinhibition to β-glucosidase,next are Al³⁺、Hg²⁺、Cu²⁺、Mg²⁺、Zn²⁺，whileFe³⁺ shows no significant effect on the enzyme activity.Fungi J-9transforms total glucosides of Centella asiatica into totalSingle glycosidases of asiatic acid in the process of liquidfermentation,then the transformation products were extracted withn-butyl alcohol and dissolved with appropriate amount of methanol,putit into silica gel chromatography column,eluted with a eluent which iscomposed of chloroform-methanol-water （10:2:0.3,V/V）,separate thetotal transformation products into A and a mixture of B and C.Put themixture of B and C onto ODS C₁₈chromatography column,eluted with aeluent which is composed of β-CD（3mg/mL）solution-methanol（1:1,V/V）to obtain purified B andC.According to the test data of specific rotation,high and low resolutionmass spectrometry（MS）, nuclear magnetic resonance（NMR）,thetransformation products A, B and C are confirmed as asiatic acid–28-O-β-D-glucopyranoside,centelloside C and chebuloside IIrespectively.Furthermore, the purity of the transformation products A, Band C are96%,97.5%and100%respectively,detected by HPLC andcalculated with area normalization method.By adding mobile phase additive β-CD,the isomers of centellosideC and chebuloside II were effectively separated,and a optimizationHPLC quantitative analysis method was build.The analysis wasperformed on a HPLC system with a Welch Materials,XB-C₁₈column（250mm×4.60mm,5μm）.The mobile phase were composed of4mmol/L β-cyclodextrin water solution（A,regulate to pH2.8withphosphoric acid）-acetonitrile（B）,graded elution（1～15min,B:26%;15～45min:B:28%）.The flow rate was1.0mL/min, the column temperature was30℃,and the detection wavelength was204nm.Asiatic acid–28-O-β-D-glucopyranoside,centelloside Cand chebuloside II can be detectedat the same time in this chromatographic conditions,andmethodological studies have shown that the method israpid,accurate,reliable,applicable to analysis the transformationproducts of total glucosides of Centella asiatica.