Influencing Factors And Mechanism Of Enzyme Production,Enzyme Activity And Kinetics Of Carbonic Anhydrase Microorganisms

Carbonic anhydrase microorganisms accelerate CO₂ hydration-induced Ca CO₃deposition.Because of its greenness,high efficiency and harmless product,it is widely used in the fields of concrete crack self-healing,efflorescence resistance and waste residue carbon fixation.The catalytic effect of Carbonic anhydrase（CA）greatly accelerates the hydration rate of CO₂,but this catalytic effect is affected by the concentration of CA and the activity of the enzyme.In this paper,the effects of five physical and chemical factors(p H,Ca²⁺、Mg²⁺、CO₃^2-and temperature)on the CA production and CA activity of mineralized microorganisms were studied;the change mechanism of microbial CA production was explained from the changes in the number of microorganisms and the ability of single bacteria to produce CA;the mechanism of CA activity change was revealed from the secondary structure level,the enzymatic activity mechanism models of concrete crack and waste residue environment were constructed respectively;the kinetics of CA enzymatic reaction and Ca CO₃ deposition under different p H、Ca²⁺and Mg²⁺concentrations were studied,analysis of changes in enzyme production,enzyme activity,enzyme catalysis and ion concentration product changes during calcium carbonate deposition.The main research contents and conclusions of this paper include the following three aspects:（1）The effect and mechanism of five physical and chemical factors on the production of microbial CA was elucidatedThe CA produced by the microorganisms cultivated under five physical and chemical factors was isolated and purified.The molecular weight of CA was 23.4 k Da as determined by mass spectrometry,and it contained very little impurities or even no impurities;the higher the p H in alkaline environment,the less the living bacteria of mineralized microorganisms and the enzyme production of single bacteria,resulting in the less CA production;when p H=8,the optimum temperature for the production of CA by microorganisms is 30℃;At 30℃and p H=8,the optimum concentrations of Ca²⁺,Mg²⁺and CO₃^2-for the production of CA by microorganisms were 40,80 and 60 m M,respectively.The above physical and chemical factors change CA production from the number of viable microorganisms and the ability of single bacteria to produce CA,that is,the less viable bacteria and the poorer the ability of single bacteria to produce CA,the lower the CA production of microorganisms.（2）The effect and mechanism of five physical and chemical factors on the activity of CA was revealedBased on the isolated and purified CA,the activity of CA in different p H,Ca²⁺、Mg²⁺、CO₃^2-concentration and temperature was determined by esterase method;the CA functional group was analyzed by FT-IR,and the secondary structure of CA was calculated by Omnic and Peakfit using the FT-IR spectrum,based on the change of the secondary structure to analyze the mechanism of CA activity change.The research shows that the p H=8 when the CA activity is the highest,and the optimal temperature is 30℃,but when the p H=13,the optimal temperature rises to 40℃;at 30℃and p H=8,the optimal concentrations of Ca²⁺,Mg²⁺and CO₃^2-were 20 m M,80 m M and 40 m M,respectively.The changes of the above five physical and chemical factors changed the structural proportions ofα-helix,β-sheet,β-turn and random coil in the CA,thus changing the order of CA structure;the increase ofα-helix andβ-sheet and the decrease ofβ-turn and random coil structure resulted in the improvement of the structural order of CA.Then,the catalytic active center of the enzyme molecule has a more stringent and more stable spatial structure due to the protection of the substrate,so the CA activity is also increased accordingly.（3）The effects of p H,Ca²⁺and Mg²⁺on the kinetics of CA enzymatic reaction and Ca CO₃ deposition were investigatedThe enzymatic reaction rates of CA with different p H,Ca²⁺and Mg²⁺concentrations were measured under different CO₂ concentrations,and the maximum rate of enzymatic reaction V_m and Michaelis constant K_m were calculated by using Lineweaver-Burk equation;The maximum velocity V_m at 30°C is reached at p H,Ca²⁺and Mg²⁺concentrations of 9,20m M and 80 m M,which were 6.84 m M/s,9.56 m M/s and 12.96 m M/s,respectively,while K_mwas the minimum,indicating that the tighter the binding of CO₂ to the CA active center,the faster the reaction rate.In addition,the effects of the above factors on the p H changes and reaction rate constants during the process of CA-catalyzed Ca CO₃ deposition were studied by NH₄HCO₃ gas diffusion system;due to the process of CO₂ hydration,sediment acidification and ammonia dissolution in the reaction process,in the reaction system with initial p H≤9,the p H of the solution first decreased and then increased,and when the initial p H>9,the p H decreased and then gradually tended to balance;when the maximum apparent deposition rate constant k of Ca CO₃ appears at 30℃,the initial p H、Ca²⁺and Mg²⁺concentrations of the system were 8,60 m M and 80 m M,and k were 5.12×10^-2/min,9.24×10^-2/min,and 10.74×10^-2/min,respectively.