Synthesis Of ?-arbutin Using Immobilized Substrate And Cells

?-Arbutin(?-Ar)is a safe and high-effective whitening product,it plays the role of whitening via inhibiting tyrosinase activity to block melanin synthesis.At present,the main production methods are microbial and enzymatic transformation catalyzing one molecule of sucrose and hydroquinone to synthesize ?-Ar.However due to the cytotoxicity of the substrate HQ,when its concentration is too high,it will cause apoptosis and reduce ?-Ar yield.In this project,a reaction system for double immobilization of substrate and cells was constructed.On the one hand,HQ was immobilized on magnetic chitosan(Fe3O4@CTS)microspheres carrier,and was slowly released to reaction system for continuous transformation.After the reaction,the carrier can be quickly separated by applying a magnetic field.On the other hand the methods of adsorption and embedding were used to prepare immobilized cells which could be used repeatedly.The main research contents are as follows:(1)Preparation of Fe3O4@CTS microspheres and its loading performance of HQ:Fe3O4 nanoparticles with a diameter of 30-40 nm were prepared by the oxidative hydrothermal method,and then Fe3O4@CTS microspheres were prepared by the emulsion crosslinking method.The characteristic absorption peaks of Schiff base bond(C=N)and Fe-O bond by FTIR were observed proving that Fe3O4@CTS microspheres were successfully prepared.The magnetic responsiveness of Fe3O4 nanoparticles and Fe3O4@CTS microspheres was analyzed by VSM.The result showed that magnetic responsiveness of Fe3O4@CTS microspheres is much lower than Fe3O4 nanoparticles but still had good magnetization indicating that it could be separated quickly by external magnetic field.The factors affecting the yield and HQ loading efficiency of Fe3O4@CTS microspheres during the emulsion crosslinking process were investigated.The results showed that the loading performance of Fe3O4@CTS microspheres were best under the conditions with 6 mL of 25%glutaraldehyde,8 mL of Span-80 and 1200 rpm of rotating speed.The loading amount of HQ on Fe3O4@CTS microspheres could reach 300-320 mg HQ/g Fe3O4@CTS microspheres under the conditions with 8 g/L of initial HQ concentration,25? of temperature,pH value of 7 and 24 h of adsorption time.(2)Preparation and catalytic activity of immobilized cells by adsorption method:First,the diatomite carrier 545 with the largest adsorption capacity for BT-112 was selected from a variety of carriers and then the adsorption conditions were explored.The results showed that the efficiency of oscillatory adsorption is much higher than static adsorption.The maximum adsorption amount could reach 112.95 mg cells/g diatomite carrier 545 under the conditions with 1:15 of the solid-liquid ratio,OD660 value 0.94 of initial cell concentration,28? of shaker temperature,100 rpm of shaker rotating speed and 2 h of the adsorption time.And the catalytic activity could reach 36.20 mg?-Ar/g immobilized cells.(3)Preparation and catalytic activity of calcium alginate(CA)immobilized cells:The factors affecting the catalytic activity of immobilized cells during the preparation of calcium alginate immobilized cells were studied.The reaction results,which was conducted in phosphate buffer saline(PBS),showed that HQ conversion rate could reach 45.20%under the preparation conditions of gel beads with 0.2%of CaCl2 concentration,3%of sodium alginate(S A)concentration,1.8 mm of gel bead diameter,OD660 value 0.372 of cell concentration and 16 h of solidification time.In the comparison experiment of culture medium and PBS as the reaction solution respectively,the former could greatly improve the substrate conversion rate,and the maximum value could reach 78.12%,which is 73.72%higher than the value 44.97%of the latter.(4)Synthesis of ?-Ar using immobilized substrate and cells:Firstly,the catalytic activity of CA immobilized cells and diatomite carrier 545 immobilized cells were compared.The results showed that CA immobilized cells had a better activity.The effects of immobilized HQ concentration,reaction temperature,shaker rotating speed and reusability of immobilized cells on ?-Ar yield were studied.The results showed that the highest ?-Ar yield could reach 8.33 g/L,which is 2.05 times of free HQ reaction group,under the conditions with 30 g/L of immobilized HQ,30? of temperature and 160 rpm of shaker rotating speed.Through the study of CA immobilized cells service life,the results showed that after being repeatedly used 5 times,the yield of ?-Ar could still reach 3.99 g/L,which effectively reduced the cost of production.