Preparation Of The Immobilized Protease Based On Metal Affinity And Its Catalytic Performance Investigation

Proteases are important industrial and commercial enzymes,which have been widely used in food,detergent,pharmacy,cosmetics,leather,chemistry,waste water treatment,etc.However,the free form of protease is associated with the issues including the difficulties in recovery and reuse,poor stability,self-hydrolysis of enzyme molecules and self-aggregation of protein molecules,which greatly hinder their industrial application.Such shortcomings can be effectively improved by the immobilized of protease for the industrial-scale enzymatic applications.Immobilized protease is easy to recycle and reuse,but the traditional immobilization methods cannot prevent the leakage of enzyme molecules and the activity loss of protease.The affinity coordination of metal and protein is stronger than physical force and weaker than chemical force,which can effectively avoid the leakage of enzyme molecules and the loss of activity through stabilizing the configuration of the active center of enzyme molecules.Immobilization of protease by metal affinity is an efficient and green immobilization strategy.In this dissertation,two different immobilization approaches were proposed based on the affinity coordination of metal and protein,one based on the immobilized metal affinity chromatography（IMAC）and the other is organic-inorganic hybrid nanoflowers.Immobilization of protease on porous carriers or nanoflowers could avoid mutual recognition of protease and provide a good microenvironment for protease.Alcalase,one of the Alkaline proteases,was immobilized on hollow mesoporous silica spheres（HMSS）by an IMAC-based immobilization method.Nanoflowers were prepared by using the principle of hybrid nanoflowers.Moreover,the catalytic performance of immobilized Alcalase and nanoflowers was investigated.1.Hollow mesoporous silica spheres（HMSS）were prepared and surface functionalized by triamino silane coupling agent.Then,the modification support was chelated with metal ions to obtain the carrier of HMSS-3N-Mⁿ⁺for the immobilized enzyme.Finally,Alcalase was immobilized through metal affinity coordination(Alcalase@HMSS-3N-Mⁿ⁺).The properties of HMSS,HMSS-3N and Alcalase@HMSS-3N-Mⁿ⁺were characterized by SEM,TEM,TEM-Mapping,BET,TGA,FT-IR,and XPS.The results show that the prepared HMSS was monodisperse spherical particles with uniform size distribution and hollow structure.The results of TEM-Mapping,FT-IR,and XPS confirmed that the successful chelation of metal ions and the immobilization of Alcalase.The p H stability,temperature stability,enzymatic kinetics,and reusability of immobilized Alcalase were investigated.Compared with the free Alcalase,immobilized Alcalase with metal affinity coordination could generally improve the p H and temperature stability of Alcalase,especially the high-temperature resistance of catalyst.Immobilized Alcalase could partially recover its initial activity after high-temperature incubation,while the free Alcalase was totally inactivated.Particularly,Alcalase@HMSS-3N-Ni²⁺could even maintain 90%of initial activity after 150 min incubation at 85℃.The immobilized enzyme has good operational stability,the overall residual activity of the Alcalase@HMSS-3N-Mⁿ⁺were higher than 40%of their original activity after five consecutive operations and Alcalase@HMSS-3N-Ni²⁺exhibited the highest residual activity of 75%after 5 times of reuse.Immobilized protease also showed higher substrate affinity and catalytic efficiency.The optimum conditions of enzymatic hydrolysis were determined as follows:50℃,p H 10(Alacalase@HMSS-3N-Fe³⁺is p H 11).2.The hydrolysis reactions of soybean protein isolate（SPI）from plant sources and Whey Protein Isolation（WPI）from animal sources were used as model reactions.The hydrolysis ability of both immobilized Alcalase and the free enzyme were characterized at the optimal reaction conditions.The polypeptide content,molecular weight distribution of polypeptide,and antioxidant activity of hydrolysate were investigated and evaluated.The results showed that the molecular weight distribution of hydrolysate of immobilized enzyme and the free enzyme was similar,which indicated that the active center of Alcalase remained unchanged after metal affinity coordination immobilization.Furthermore,SPI and WPI have been hydrolyzed,and the HPLC spectrum shows that the peptides below 1500 Da account for88%and 87%of the hydrolysate respectively with 45%and 40%of them being MW<500Da.The antioxidant activity of SPI and WPI hydrolysate was characterized,the results showed that the low molecular weight polypeptides prepared by different metal ions immobilized Alcalase had good biological activity.The biological activity was consistent with the degree of protein hydrolysis and the polypeptide content of hydrolysate.These results indicated that the prepared metal affinity immobilized Alcalase can efficiently prepare bioactive peptides.C ompared with the hydrolysis reaction of SPI and WPI,the immobilized Alcalase showed high hydrolysis efficiency for low solubility proteins.3.Organic-inorganic hybrid nanoflowers of AY-10@AXH-HNFS were successfully prepared by the coordination between Cu²⁺and protein molecules with phosphate buffer,copper sulfate solution,protease AXH,and AY-10 as raw materials.The effects of Cu²⁺concentration,immobilization time,enzyme concentration,and AXH/AY-10 ratio on the preparation of AY-10@AXH-HNFs were investigated.The optimal preparation conditions were determined as follows:4 m M Cu²⁺concentration,single enzyme immobilization for 24hours,the total concentration of 0.6 mg/m L,and AXH/AY-10 ratio of 1/1.5.The AY-10@AXH-HNFs were characterized by SEM,TEM,SEM-EDS,BET,TGA,FT-IR,XPS,and Confocal laser scanning microscope.The results confirmed that AXH and AY-10 was coordinated and hybridized with Cu²⁺successively to prepare the co-hybrid nanoflowers,which contained about 14%enzyme molecules.AY-10@AXH-HNFs had a good pore size distribution with an average pore size of 60.2 nm and the desorption curve showed that there was a mesoporous structure in nano-flowers.Multi-stage pores were beneficial to substrate mass transfer and enzymatic catalysis.The activities of AY-10@AXH-HNFs and free enzyme were investigated with BAEE as substrate.The activity of AY-10@AXH-HNFs was much higher than that of free enzyme,reaching 4.1 times free AXH and 9.6 times free AY-10.The effects of p H and temperature of free enzyme and AY-10@AXH-HNFs were compared.The results showed that the activity of prepared AY-10@AXH-HNFs was higher than that of free enzyme under various conditions.After five cycles,the catalytic activity of AY-10@AXH-HNFs is still higher than that of free enzyme,and it has good reusability.