Culture Optimization And Immobilization Of Recombinant Aspergillus Niger GZUF36 Intracellular Esterase

Esterase is a kind of?/?hydrolase,which can not only synthesize esters but also hydrolyze ester bonds.It has good industrial application value in dairy baking,chiral drug research,washing industry,pesticide insecticide et al.The culture of enzyme is easy to be interfered by external factors,and the free esterase has some disadvantages,such as poor stability,short storage time,low-reuse rate.Therefore,on the basis of the recombinant Aspergillus niger lactonase(rINANE)obtained in the laboratory,this study first explored the external factors affecting the expression of rINANE in Pichia pastoris for obtaining high activity of rINANE.Then,the magnetic ferroferric oxide coated with polydopamine(Fe₃O₄@PDA)was used as the adsorption carrier,and the immobilization technology of adsorption-precipitation-crosslinking was used to study the immobilization of rINANE.Through the characterization of enzyme structure before and after immobilization,the change of enzyme structure was explained.Finally,the properties of immobilized enzyme and free enzyme were compared to further illustrate the changes of enzyme properties caused by structural changes.These studies can provide a basis for the study of the structure-activity relationship of rINANE and its large-scale use in industry.The main research contents and results are as follows:(1)The p H,temperature,methanol dosage,initial OD₆₀₀,centrifugal speed and induction time during induction were studied.It was found that induction time is the most important factor affecting the activity of rINANE,followed by p H and temperature.Through uniform design and programming,the optimal culture conditions were obtained as follows:BMMY medium p H=6.0,the temperature of induction culture is 25?,methanol induction dosage of 1.5%,induction initial OD₆₀₀=1.0,centrifugal speed of 4000 r/min after induction,induction time of 120 h.Under this condition,the enzyme activity of rINANE is 1.925±0.053 U/ml,which is about 3 times higher than that under the initial culture condition.(2)With Fe₃O₄@PDA as the adsorption carrier,rINANE was immobilized by the technology of adsorption-precipitation-crosslinking.The effects of different immobilization conditions on the activity of rINANE were studied.When the solid-liquid ratio of Fe₃O₄@PDA carrier to rINANE is 45:1 mg/m L,the adsorption time is60 min,the optimal amount of acetone is 4 m L/m L,pectin is 3?L/m L,and the crosslinking time is 1 h,the enzyme activity recovery and protein loading of immobilized rINANE are 104.40±1.14%and 72.72±1.01 mg/g,respectively.(3)For exploring the success of immobilization and the change of enzyme structure before and after immobilization,the free rINANE and samples during enzyme immobilization were characterized by scanning electron microscope(SEM),Fourier transform infrared(FTIR),X-ray diffraction(XRD),zeta potential and sample magnetic intensity(VSM).SEM results showed that rINANE has been successfully immobilized.Through the adsorption-precipitation-crosslinking immobilization technology,the enzyme molecules are closely linked,forming a network structure with holes,and the rigid structure is enhanced,which can lock the substrate and improve the enzyme activity.After immobilization,the relative content of?-helix decreased by 8.73%and the?-fold increased by 39.74%,which confirmed that the rigid structure of rINANE is enhanced after immobilization.XRD results showed that the crystal structure of the carrier did not change after immobilization.The zeta potential absolute value of immobilized rINANE is 27.42±0.27 m V,which is higher than rINANE,indicating that the stability of immobilized rINANE solution is better than rINANE.The saturation magnetic intensity of immobilized rINANE is 75.193 emu/g,which has strong magnetism,and the enzyme structure remains stable under the action of magnetism.(4)The enzymatic properties of rINANE before and after immobilization were explored.The results showed that the optimal temperature of immobilized enzyme(40?)is 5?higher than that of free enzyme,and the optimal p H was also transferred to alkaline condition by one unit,which is 9.0.The immobilized enzyme could maintain more than 70%of the enzyme activity after 12 h treatment at 20?-45?,which is higher than the free enzyme.It could still maintain more than 80%of the enzyme activity in the range of p H 6.0-10.0.With the addition of surfactant,the free enzyme activity is significantly increased,while the immobilized enzyme activity remained in a relatively stable range.Similarly,the tolerance of immobilized enzyme to Cu²⁺,Zn²⁺,K⁺,Na⁺,Mg²⁺,Ca²⁺,Fe³⁺and organic solvents is higher than free enzyme.By comparing the kinetic parameters of free enzyme and immobilized enzyme,it was found that the Michaelis constant km of immobilized enzyme is 16.55 g/L,which is higher than that of free enzyme(10.86 g/L),indicating that the affinity of immobilized enzyme to substrate decreased.After 45 days of storage,the immobilized enzyme still maintained 86.6±2.1%of the enzyme activity.Repeat the operation 8 times,and there is still 55.2±1.1%enzyme activity,which has good storage and operational stability.These studies provide a certain experimental basis for the change of rINANE basic properties,and lay a foundation for the structure-activity analysis of rINANE and the low-cost recycling of immobilized rINANE in industry.