Research Of Enzymatic Immobilization By Biomineralization

Among the existed catalytic types,the use of enzyme catalysis is a green,convenient and quick way.However,some disadvantages seriously limited the application of enzymes in industrial production,for example,the high cost of the enzyme,complicated preparation procedure and the inability for recycle,et al.Therefore,by immobilizing the enzyme on a specific carrier,the stability of the enzyme can be improved to a certain degree,and ensure that the enzyme can be reused.Biomineralization is an emerging immobilization method.By coprecipitation of organic components?such as proteins or amino acids?and inorganic constituents?phosphates,etc.?,biomineralization can produce a novel type of nanoparticle with complex shapes,layered structures,uniform particle sizes and high strength.In that case,scientists started to simulate the crystallization process in vitro.The nanoparticles with a flower-like structure can be produced by this crystallization technique and has been defined as?nanoflower‘with an inorganic component as skeleton.This study is mainly divided into two parts.The first part is the immobilization of lipase?CSL?with zinc phosphate?Zn₃?PO₄?₂?as carrier.The The effect of CSL concentration,preparation time,temperature,ionic strength and immobilized organisms on the shape and size of CSL-Zn₃?PO₄?₂ nanoparticles were studied in detail.During the experiments,we ensured the optimum conditions for preparing nanoflowers as follows:zinc sulfate solution?80 mg/mL,5 mL?was added dropwise to the enzyme solution?100 mL?,and the CSL concentration was 0.6 mg/mL.After stirring at room temperature?about 20??for 1 hour and resting for 2 hours,the optimal nanoflowers were obtained.Scanning electron microscopy,Energy Dispersive X-ray Spectroscopy,Transmission electron microscopy,Fourier Transform infrared spectroscopy,X-ray diffraction crystal diffraction,Elemental analysis were used to characterize the nanoflowers.With these detections,we confirmed that the nanoflowers we prepared contained both Zn₃?PO₄?₂ components and N element of CSL,which proved that CSL was indeed immobilized on the carrier,and the CSL content in the nanoflower is calculated to be about 7.2%.And then,we achieve acetylation of arbutin catalyzed by prepared nanoflowers.Compared with the free CSL,the enzyme activity of the hNFs?15.9±0.5 U?was significantly enhanced about 4.3-folds when the added enzyme content was the same.Meanwhile,the thermal stability was also greatly improved.After 10 cycles of using the nanoflowers,enzyme activity still retained about 95%of the initial;after 20 days of storage,about 92%of initial activity was retained,it can be seen that the stability of immobilized enzyme is very high.With the studying of the preparation and catalysis of CSL-Zn₃?PO₄?₂nanoflowers,we can't help thinking that if we could find a metal-dependent enzyme,and combine the enzyme with the metal,the advantages of conventional immobilization could be achieved,and the enzyme activity could be greatly improved at the same time.Therefore,in the second part of this study,we selected a metal-dependent enzyme named D-psicose-3-epimerase?DPEase?.The purity of DPEase was over 90%after purification,and the molecular weight was about 40 kDa.We studied various physical and chemical properties of free DPEase and found that DPEase is a Co²⁺,Mn²⁺-dependent enzyme.The enzyme activity is maximized when100?M Co²⁺is added;the optimum pH is 8.0;the optimum temperature is 60?,while the thermal stability and pH stability are poor.In order to solve these problems described above,we adopted the method of biomineralization.Since DPEase is a Co²⁺-dependent enzyme,cobalt phosphate could be an optimal choice as an inorganic carrier,which can improve the stability of DPEase after immobilization,and further enhance the enzyme activity by the activation of Co²⁺.It is also the first report about immobilization of DPEase on Co₃?PO₄?₂ by biomineralization.Through in-depth study of the preparation of DPEase-Co₃?PO₄?₂ and influencing factors related,We summarized the optimal reaction conditions as followed:2mg DPEase was mixed with phosphate?pH 7.4,50.0 mM?,and 10.0?l CoSO4?1.0 M?was added.After stirring at 4?for 48 h,the resulting precipitate was the form of nanoflower.Scanning electron microscopy,Energy Dispersive X-ray Spectroscopy detection,Fourier Transform infrared spectroscopy detection and Elemental analysis were used to characterize the prepared nanoparticle.The amide I/II bands?DPEase?and Co/P elements?Co₃?PO₄?₂?were identified.The DPEase content of nanoflowers was calculated to be about 12%.In enzymology studies,the catalytic activity of the prepared nanoflowers?36.2±0.5 U/mg?was 7.2 times higher than that of free DPEase?5.0±0.2 U/mg?.For the significant increase of enzyme activity,we performed a detailed analysis:Since DPEase is strictly Co²⁺-dependent,in the formation of nanoflowers,Co²⁺can bind to specific functional sites of DPEase and activate DPEase through Allosteric Effect.In addition,Co²⁺and DPEase are restrained into the limited space of nanoflowers,Co²⁺is very close to DPEase,in that way DPEase can be easily activated.Therefore,most of DPEase active forms can be enriched or?locked?during the immobilization process,which may be a reasonable explanation for the high increased activity of the nanoflowers.In this study,we used intelligent biomineralization methods to prepare CSL-Zn₃?PO₄?₂ and DPEase-Co₃?PO₄?₂ nanoflowers.The new method combines the carrier preparation with the enzyme immobilization perfectly,which greatly simplifies the immobilization process.In addition,this biomineralization method can significantly increase the enzyme activity and greatly improve enzyme stability,indicating that this new biomineralization method can be widely applied to the immobilized on the enzyme,especially a metal-dependent enzyme.