Design Of Immobilized Enzyme Carriers Based On Multi-parameter Collaboration In Microenvironment And Its Application

Immobilized enzyme has attracted the attention of many researchers as one of the intersecting research point in many fields such as materials,biology,and chemistry.With the deepening of immobilized enzymes research,the immobilization enzymes is undergoing the evolution from carrier selection to carrier design.The microenvironment of immobilized enzymes directly determines the activity,stability and selectivity of the immobilized enzymes.Studies on the effects of single factors of microenvironment such as pore diameter,hydrophilicity/hydrophobicity,Zeta potential,specific surface area,and pH on the activity of immobilized enzymes have been reported.However,there are few reports on the synergistic effects of multiple parameters on enzyme activity in the microenvironment.Therefore,the study of the synergistic effect of the immobilized enzymes activity on the immobilized enzymes microenvironment is of necessity.In addition,the study has academic research value and potential application value for the industrial application of the immobilized enzymes,and it provides the basis for the design of the immobilized enzyme carrier.Based on the statistical analysis of correlation analysis,7?-hydroxysteroid dehydrogenase?7?-HSDH?and 7?-hydroxysteroid dehydrogenase?7?-HSDH?were used as model enzymes.The relationship between the pore diameter,hydrophilicity/hydrophobicity,surface charge,specific surface area and the activity of enzyme were investigated.The multiple regression models of immobilized enzymes microenvironment and enzyme activities was constructed for exploring synergistic effects of microenvironments on the activity of immobilized enzymes.Therefore,the study provide the theoretical basis for the design of immobilized enzymes carriers.On this basis,a series of immobilized enzymes carriers were designed on the basis of epoxy resins.The 7?-HSDH and 7?-HSDH were co-immobilized for the in situ biotransformation of chicken gall powder solution.Thus,the application of immobilized enzyme carriers designed on multiple parameters of the microenvironment was expanded.The main research contents and results are as follows:First,based on the statistical methods of correlation analysis,the relationship between immobilized enzyme microenvironment and enzyme activity were studied,and a multiple regression model of immobilized enzyme microenvironment and enzyme activity were established.Zeta potential,hydrophilicity/hydrophobicity,pore diameter and specific surface area were characterized about 21 kinds of different resins.7?-HSDH and 7?-HSDH were used as model enzymes to immobilize on different microenvironment carriers.The specific activities of immobilized 7?-HSDH and7?-HSDH were correlated with the microenvironment parameters of the carriers,and the degree of correlation was evaluated according to the absolute value of the correlation coefficient.The result showed that the most significant influence on the activity of immobilized 7?-HSDH and 7?-HSDH was the pore size of the carriers?correlation coefficients were-0.8202,-0.8603,respectively?,followed by the zeta potentialof the carriers?correlation coefficient was 0.5992,0.5563,respectively?.The medium hydrophobic?water contact angle was 50°?carrier provides the suitable microenvironment for immobilized 7?-HSDH and 7?-HSDH to exhibit the highest activity.The multiple regression model for the specific activity of immobilized7?-HSDH and 7?-HSDH and four microenvironment parameters was established.The model is as follows.y₁=0.008448x₁+0.0003340 x₂-0.008708x₃+0.000227x₄+0.3306;y₂=0.012100x₁+0.0005980x₂-0.01655x₃-0.00002700x₄+0.6645,y₁ and y₂ are the specific activities of immobilized 7?-HSDH and 7?-HSDH,respectively;x₁ is the zeta potential of the carrier;x₂ is the contact angle of water;x₃ is the pore size of the carrier;x₄ is the specific surface area of the carrier.The external verification correlation coefficient of the model?R²?are 0.7154 and 0.7532,respectively.The two models have achieved satisfactory predictive results and can reflect the synergistic effects of several parameters of the immobilized enzyme microenvironment on the activity of immobilized enzymes,so that provide reliable basis for the design of immobilized enzyme carriers.Secondly,based on the above correlation analysis and multiple regression model conclusions,a series of immobilized enzyme carriers were designed based on different quality percentage concentrations of chitosan-modified epoxy resins.These carriers have medium hydrophobic and different crowding and charge conditions of the microenvironment.Multiple regression models were used to predict the activity of immobilized enzyme.The effects of the immobilized enzyme microenvironment on the crowding degree and the charge on the properties of the immobilized enzyme were investigated.The result showed that the activity of immobilized enzymepredicted by multiple regression model was consistent with the experimental value.The activities and thermal stabilities of immobilized 7?-HSDH and 7?-HSDH can be improved by manipulating the crowding and surface charge effects.As the pore size of the carriers decreases,that is,the degree of crowding of the immobilized enzyme microenvironment increases,the activity of the immobilized enzyme is effectively enhanced.The increase in the activity of the immobilized enzyme results in an effective increase in the catalytic efficiency of the immobilized double enzyme.Changing the degree of crowding of the immobilized enzyme microenvironment also enhances the thermal stability and cycling stability of the immobilized enzyme.After 7 successive cycles,the catalytic efficiency of the EP-0.5-C immobilized double enzyme was small decreased?TCDCA conversion rate decreased from 85.45±0.36%to 84.32±0.55%,and the TUDCA yield decreased from 55.02±2.07%to 51.54±0.67%?.The results are consistent with the above-mentioned correlation analysis and model.These findings suggest that crowding and charge of the microenvironment can significantly improve the properties of the immobilized enzyme.Then,the changes of microenvironment can not only change the properties of the immobilized enzyme,but also change the properties of the immobilized enzyme carrier.The adsorption behaviors of EP-0.5-C for TCDCA and TUDCA were further explored,and compared with epoxy resins.The adsorption capacity of EP-0.5-C for TUDCA and TCDCA were lower than epoxy resin.EP-0.5-C has superior mechanical properties than chitosan microspheres.The use of chitosan modified epoxy resin to change the microenvironment of the immobilized enzyme carrier can improve the performance of the carrier.Finally,in transformation of complex substrate chicken bill powder solution with7?-,7?-HSDH co-immobilized on EP-0.5-C was used to prepare the bear bill powder analogue.The reaction conditions were optimized.After 9 times of reuse,the conversion rate of TCDCA in chicken bill powder remained at 69.82±2.1%,and the yield of TUDCA remained at 46.77±2%.The chitosan microspheres were used as the carrier to immobilize 7?-,7?-HSDH in the past studies.After four consecutive conversions,the conversion rate of TDCCA was lower than 40%,and the yield of TUDCA was lower than 20%.The shows that the co-immobilized 7?-,7?-HSDH on EP-0.5-C has good reusability.The product components showed that the most of the bile acid component TCDCA was converted to TUDCA,some non-bile acid components were retained.The purpose of the in-situ bioconversion is achieved and the main components of the reaction product are similar to the natural bear bill powder,provide reliable basis for the design of immobilized enzyme carriers.