Self-assembled Synthesis Of 3D Nanocomposites With Alkaline Protease And Metal Ions

Enzyme is the core of biocatalysis,however,the harsh reaction conditions often lead to the low stability,loss of enzyme activity or inactivation during the actual industrial catalytic reaction,which strictly hamper its applications.In order to overcome these difficulties,the methods of enzyme immobilization have been put forward and developed.Enzyme immobilization is a well-known essential process to improve enzyme stability and enable the industrial reuse of enzymes for more reaction cycles.But several weaknesses exist after immobilization for reducing the enzyme activity.In past decades,the foundation of the nano/microsized immobilization method not only maintaining the stability of enzyme,but also improving the catalytic activity effectively.However,the process of immobilization is complicated and costly,which hard to control.Therefore,it is extremely important to establish a new fast,simple and economical immobilized method.Firstly,Cu²⁺was added to PBS that contained alkaline protease to synthesize 3D nanocomposites with peony-like structure by self-assembly method.The inorganic carrier of this material is Cu₃?PO₄?₂·3H₂O,which was determined by FT-IR,XRD and EDS.By optimizing the synthetic conditions of enzyme concentration and pH of nanocomposites,at25 ^oC,when the enzyme concentration was 0.1 mg/mL and the pH value was 8.0,the alkaline protease-Cu₃?PO₄?₂·3H₂O nanocomposites exhibited extremely high enzyme activity?1220.27 U/mg?that almost 13 times higher than free enzyme?93.70 U/mg?.Moreover,after six reaction cycles,the enzymatic activity of this material was 184.12 U/mg,which was still much higher than that of free enzyme.Then,Zn²⁺was added to PBS that contained alkaline protease to synthesize 3D nanocomposites with daisy-like structure by self-assembly method.The inorganic carrier of this material is Zn₃?PO₄?₂·4H₂O,which was determined by FT-IR,XRD and EDS.By changing the preparation conditions of enzyme concentration and pH of nanocomposites,the optimal enzyme activity of the alkaline protease-Zn₃?PO₄?₂·4H₂O nanocomposites was241.04 U/mg that almost 2.6 times higher than free enzyme,which were synthesized at 25 ^oC,0.25 mg/mL enzyme concentration,pH 6.0.In addition,by evaluating the reuse property ofthe alkaline protease-Zn₃?PO₄?₂·4H₂O nanocomposites,the enzyme activity decreased significantly after three reaction cycles.And SEM pictures were fully demonstrated the regular 3D flower structure was an important condition for maintaining the higher enzyme activity of the nanocomposites.According to the above studies,it was found that the morphology,size,actual enzyme content,and enzyme activity of alkaline protease-Cu₃?PO₄?₂·3H₂O or alkaline protease-Zn₃?PO₄?₂·4H₂O nanocomposites were affected by the initial enzyme concentration significantly.This study has a certain role in promoting the wide application of enzyme in biocatalysis,biosensing,medicine and energy.