Synthesis Of Cobalt Phosphate In Different Solvent Systems And Its Application In The Immobilization Of β-glucosidase/B Romelain By Affinity Adsorption

Compared with chemical catalysts,biocatalysts had the advantages of high catalytic efficiency,mild reaction conditions,strong specificity and environmental friendliness.However,the low stability and high application costs of biocatalysts had seriously hindered the industrial application of biocatalysts.The immobilization of enzyme can improve the stability of enzyme,and reduce the application costs of enzyme through recycling.Therefore,the research of enzyme immobilization methods and immobilization carriers was an important direction in this field.For the physical adsorption method of immobilized enzyme,how to improve the adsorption strength of enzyme on the surface of carrier material was an urgent problem.In this project,a metal cobalt phosphate carrier material was prepared in two solvent systems,using His-taggedβ-glucosidase and bromelain as model enzymes.The immobilized enzyme was based on the coordination between the metal ion and the electron-donating group on the amino acid residues such as histidine/cysteine/tryptophan on the target enzyme to strengthen the physical adsorption between the enzyme and the material,thereby improve the stability of immobilized enzymes.The main research contents were as follows:（1）Using water as the reaction solvent,the cobalt phosphate carrier material was prepared under normal temperature and pressure,and used for the one-step separation and immobilization of His-tagged recombinantβ-glucosidase（referred to as GLH）in cell lysates.The effects of Co²⁺concentration,preparation temperature of Co₃（PO₄）₂,immobilization time,immobilization temperature and mass ratio of GLH were investigated;At the same time,the thermal stability,storage stability and cycle stability of immobilized enzyme were investigated in detail.Under optimal conditions,activity recovery and relative activity of Co₃（PO₄）₂@GLH reached 82.1%and 1.11,and the maximum specific enzyme activity was 7887.1 U/g.Cyclic stability and storage stability of immobilized GLH had been significantly improved,and 71.7%of the initial enzyme activity could still be retained after 8 times of recycling under 40^°C and the original enzyme activity was still 72.4%after 30 days storage at 4^°C.（2）The deep eutectic solvent（DES）was used as the solvent and template,and the cobalt phosphate carrier material was prepared by ionic thermal synthesis and used for the immobilization of bromelain.The physical adsorption of bromelain was enhanced by the coordination of Co²⁺with histidine,cysteine and tryptophan residues on the target enzyme.The effects of cobalt salts species,DES species and the ratio of Co²⁺to PO₄^3-（referred to as Co/P）were investigated to explore the morphology of cobalt phosphate and the performance of immobilized enzyme.Under the optimized conditions,the activity recovery and immobilization rate of immobilized Co-P@bromelain were 65.9%and 65.3%,respectively,and the maximum specific activity was 71244 U/g.The immobilized bromelain showed good cycle stability and storage stability.The enzyme activity retention rate was 55.2%after being recycled for 8 times at 37^°C,and the original enzyme activity was still 92.4%after 30 days storage at 4^°C.