Preparation And Properties Of Immobilized Naringinase And Naringenin Preparation By Enzymolysis

Naringinase,which can specifically hydrolyze naringin into naringenin,is an enzyme complex consisting of?-L-rhamnosidase?EC 3.2.1.40?and?-D-glucosidase?EC 3.2.1.21?.Naringinase is widely exist in nature,it has many important applications in food and pharmaceutical industries.Moreover,the recovery yield and reusability of free enzymes as industrial catalysts are quite limited and,hence,attention has been paid to enzyme immobilization.In this work,naringinase obtained from Aspergillus aculeatus JMUdb058 under solid-state fermentation,was immobilized into matrix consisting of polyvinyl alcohol?PVA?and sodium alginate which were crosslinked with glutaraldehyde.The enzyme activity of naringinase and?-rhamnosidase was analysed by HPLC method.The optimal immobilized conditions were achieved with 11%PVA and 0.5%sodium alginate at pH 4,1%glutaraldehyde for 0.5 h,141 U of enzyme load of per milliliter matrix,adsorption time of 3 h,4%boric acid and 1%CaCl₂,respectively.In addition,the research for immobilized enzymology properties showed that the optimum temperature,pH and The Michaelis constant?K_m?of immobilized enzyme was higher than that of free form.Compared with free enzyme,pH stability revealed no change by immobilization,and instead,temperature stability and of immobilized enzyme was decreased5oC.In this study,the dynamic conversion process of naringin into naringenin catalyzed by immobilized naringinase was investigated and the DNS method was employed to evaluate the effects of substrate concentration,enzyme dosage,pH,temperature,vibration rate on hydrolysis of naringin.The results showed that the optimal conditions of naringin bioconversion were temperature 50oC,pH 5.0,enzyme dosage 3.5 U/mL,substrate concentration 0.2 g/100 mL,and speed oscillation 120 r/min.Under the optimum conditions,the conversion rate of naringin could reach 93.3%.The double reciprocal plot of Lineweaver-Burk fit equation is y=0.6821x+0.5784,the values of K_m and V_max were 1.18 g/L and 1.73 mg/min.The generated narinenin was extracted from the hydrolysate and authenticated by thin-layer chromatography,and the naringenin recovery was calculated by 86.8%.The magnetic nanoparticles can be easily recovered by magnetic field will have potential application in industry,so naringinase immobilized by it was studied.Magnetic Fe₃O₄nanoparticles were prepared by chemical coprecipitation method.The synthesized materials were characterized by TEM,SEM,FTIR,XRD,pecific surface area,Zeta potential distribution,Hysteresis loop,TGA and so on.Meanwhile,immobilized enzymology properties was studied.Subsequently,the immobilization process was investigated by examining enzyme concentration,glutaraldehyde concentration,immobilization time,temperature,pH and speed oscillation.The results showed that the size of Fe₃O₄ was about 15 nm,it's size was increased by immobilization.And the pecific surface area,zeta,saturation magnetisation value of were decreased by immobilization.The content of Fe₃O₄ was up to 62.1%in naringinase-Fe₃O₄nanoparticles.Meanwhile,as a result,it could gain an immobilized protein load reach as high as53%and the activity recovery was up to 32%while 0.35 mg/mL enzyme and 3%glutaraldehyde were mixed at pH 5.0 and 28oC for 4 h.In addition,the optimum temperature and pH of naringinase revealed no change by immobilization.The naringinase-Fe₃O₄ nanoparticles exhibited a better pH stability than free naringinase,and instead,temperature stability of naringinase was decreased by immobilization.The Michaelis constant?K_m?showed that affinity of immobilized enzyme for binding substrate was higher than free enzyme.And the residual activity was about 83%after 8 cycles.After 60 days stores in 4oC,the immobilized naringinase still retained over 78%of its original activity.In this paper,naringinase was immobilized into matrix consisting of polyvinyl alcohol?PVA?-sodium alginate and magnetic nanoparticles.Meanwhile,characterization of immobilized and free naringinase was studied.As a result,the application of immobilization technology could enlarge the applied scope of naringinase,and it also could improve specificity,activity and stability of enzymes.Subsequently,the dynamic conversion process of naringin into naringenin catalyzed by immobilized naringinase was investigated in order to modify naringin,reduce the cost of production of naringenin,and simplify purification processes.