Preparation Of Enzyme@Inorganic Hybrid Nanoflowers And Its Catalytic Synthesis Of Fine Chemicals

Fine chemicals are a class of chemical substances with specific functions and high usability,which are widely used in food,medicine,catalyst and other fields,and their synthesis by traditional methods is seldom environmentally friendly and suitable for sustainable development.On the contrary,the biocatalysis using enzymes,can be carried out with high specificity and efficiency under mild conditions and fulfil the requirements in green chemistry and engineering.However,free enzymes have the disadvantages such as poor environmental tolerance and inability in practical applications.Enzyme immobilization is a commonly used method to tackle these barriers and improve enzyme properties.Due to the different characteristics from different type of enzymes,different immobilization method is chosen to be suitable and favorable to immobilize the given enzyme to improve enzyme loading and activity.In this work,depending on the mimetic biomimetic mineralization,enzyme protein are used to co-precipitate with phosphate to form enzyme@inorganic hybrid nanoflowers with enhanced catalytic performance and stability.The obtained enzyme@inorganic hybrid nanoflowers are used to catalyze the synthesis of fine chemicals such as 1,3-dioleic acid-2-palmitinyl glyceride and 5'-fluorodeoxyadenosine,which could further extend the scope of application of biocatalysis methods.1,3-Dioleyl-2-palmitoyl glyceride?OPO?belongs to triacylglycerol,is one of the important components of human milk fat,and thus commonly used in infant formula as an important additive.In this work,Pichia pastoris strain GS115 was selected to express and produce lipase?Thermomyces lanuginosus lipase,TLL?.Depending on biological mineralization,this lipase is co-precipitated with phosphate to synthesize TLL@hydroxyapatite nanoflowers?TLL@HAp-NFs?.After heating at 40°C for 5 h,TLL@Ca P can retain its full activity,and only 54%for free enzyme.While heating at70°C,the free enzyme activity rapidly decreased to about 23%after 1 hour,and TLL@Ca P still retained 30%of the enzyme activity,which indicates that the thermal stability was significantly improved by immobilization.Under the optimal conditions,the water content in the reaction was 2%,the molar ratio of substrate PPP/OA was 1:6,6 m L of n-hexane and lipase with content of 20 mg·m L^-1?20%?was used.After 12hours of reaction at 35°C,the content of OPO in the product can reach 50.6%,which is about 1.3 times higher than that obtained using free TLL.The prepared TLL@HAp-NFs was recycled for 5 times,and the content of OPO in the last reaction was still above90%.Therefore,this selection of the immobilization method improves the thermal stability and catalytic activity of the lipase.Additionally,calcium phosphate as the inorganic ingredient in the hybrid nanoflowers is safe for healthy infant food,and risk resulting from the monomers leaking from polymer support will not occur to these inorganics.Moreover,we also prepared fluorinase@hydroxyapatite nanoflowers?FDAS@FHAp-NFs?and catalyzed the biosynthesis of drug and radiomedicine containg fluorine using this hybrid nanoflowers.E.coli Rosetta?DE3?was selected to express the fluorinase after screening for fluorinase?FDAS?and its expression conditions.To prepare the highly stable fluorinase@fluorinated hydroxyapatite nanoflowers?FDAS@FHAp-NFs?,based on the previous work and characteristics of fluorase,potassium fluoride is used as additive to keep fluoride ion concentration of 20ppm,and expected to improve the immobilization of fluorinase in the nanoflowers.It can be seen that the petal size and shape of the nanoflowers are smaller and more regular than that of the FDAS@HAp-NFs by SEM,XRD and FT-IR characterizations.The kcat/Km value of fluorinase@FHAp-NFs is also about twice that of free enzyme,which means the enhaned catalytic efficiency after immobilization.Moreover,FDAS@FHAp-NFs still retain 62.6%of its initial activity after heating at 50°C for 8 h,and FDAS@HAp-NFs retain only 30%of its initial activity,while free enzyme is almost inactivated and no activity was detected.Therefore,using this immobilization method with fluoride as additive improved the catalytic activity and thermal stability and of FDAS@FHAp-NFs.In summary,we prepared the enzyme@inorganic hybrid nanoflowers using biomimetic strategy and based on the principle of biomineralization.While improving the thermal stability and tolerance to environment conditions of the enzyme,the obtained hybrid nanoflowers present elevated catalytic activity.This may be resulted from the confinement of the structure of the enzyme protein and the mimetic macromolecular crowding effects in the nanoflower.The effects possible increase the fold of enzyme protein chain.Moreover,calcium ions are apt to chelate with amino and carboxyl group of the amino acid residues in enzyme protein peptide chains,which stabilize the enzyme structure and elevate the catalytic activity of enzymes.Furthermore,the immobilized biocatalyst present augmented specific surface area,which facilitates the attachment of substrate on the support surface,and thus increases the catalytic activity and kinetic property.Calcium phosphate is used as inorganics composition to prepare the immobilized enzyme and the technology is energy-efficient and green,which can be utilized as an eco-friendly catalyst to produce fine chemicals.