hrvatski

Matematički model aldolne adicije katalizirane D- fruktoza-6-fosfat aldolazom, FSA A129N/A165G

Aldol addition of dihydroxyacetone to N-Cbz-3- aminopropanal catalyzed by D-fructose-6- phosphate aldolase FSA A129N/A165G from genetically modified Escherichia coli was studied. An organic solvent (co-solvent) is necessary in the reaction mixture because of the poor solubility of the substrate (N-Cbz-3- aminopropanal) in water. Therefore, the influence of organic solvents (acetonitrile, ethyl acetate, acetone, and dimethylformamide) on enzyme activity was investigated in the batch reactor. The results show that the enzyme is the most stable with ethyl acetate as co- solvent. The aim of this work was to determine the kinetics of aldol addition and to develop a mathematical model of the process in the batch reactor. Kinetics of the aldol addition can be described with a double substrate Michaelis- Menten kinetics. Kinetics of the retro-aldol reaction can be described with Michaelis-Menten kinetics with included competitive inhibition by N-Cbz-3-aminopropanal. Kinetic parameters were estimated from the experimental data. Maximum specific activities of enzyme in the forward and the backward reaction were: Vm1 = 2.860 U mg-1, Vm2 = 18.199 U mg-1, Michaelis constants KmN-Cbz-3-aminopropanal = 24.335 mmol dm-3, Kmdihydroxyacetone= 50.116 mmol dm-3 and Kmaldol = 188.948 mmol dm-3, and inhibition constant KiN-Cbz-3-aminopropanal = 0.0489 mmol dm-3. Mathematical model of the process was validated in the batch reactor. Aldol addition reaction was also carried out in a microreactor with acetonitrile and dimethylformamide as co- solvents. Enzyme activity was followed during the reaction and the operational stability decay rate constants for both co-solvents were estimated. The conversion of the substrate increases with residence time, while the enzyme activity decreases. By comparing the values of the operational stability decay rate constants (kdacetonitrile= 0.0522 min -1, kddimethylformamide = 0.1239 min-1), it can be seen that the enzyme is more stable in acetonitrile.

kinetika ; aldolna adicija ; mikroreaktor ; ko-otapalo

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