Thermokinetic Studies On The Action Of Recombinant Escherichia Coli

Recombinant Escherichia coli B1 and B2 were studied using calorimetric technique. The thermokinetic characteristics of the biological process, such as growth and metabolism, were parsed with the theory of thermokinetics and the thermochemical relationship between various conditions, such as temperature, kanamycin doses, Mg²⁺ concentrations, pH and cell concentrations, and the growth of Escherichia coli was discussed.With respect to the fact that recombinant E. coli B1 and B2 used is thermo-inducible bacteria, its growth process should be analyzed during two different phases - above or below 39℃. According to the thermal power-time curves at different temperatures, the generalized logistic equation at temperatures below 39℃and the linear growth model at temperatures above 39℃were found. In accordance with the generalized logistic equation, the growth rate constant k, the generation time G, the heat effects Q, and the thermokinetic parameters, such as the activation entropyΔS^≠, the activation free energyΔG^≠, the activation enthalpyΔH^≠and the equilibrium constant K^≠, of the process at temperatures below 39℃were calculated. Non-linear equations for k - T (E. coli B1 and B2) were established during temperatures ranging from 22℃to 37℃. The optimum growth temperature and the lowest growth temperature are 36.50℃and 4.22℃for B1 as well as 36.50℃and 4.77℃for B2. These results indicated that high temperatures promoted the growth and metabolism of recombinant E. coli B1 and B2 and had some stimulatory effects.According to the thermal power-time curves of different kanamycin concentrations, the corresponding thermokinetic equations were established and analyzed with two different theories. The results indicated that with increasing doses of kanamycin, the growth rate constant k and the maximum thermal power P_m all decreased with dissimilar speeds. The value of k reduced drastically from 0.0596 min^-1 to 0.0387 min^-1 when the kanamycin concentration varied from 0-2.5μg/ml. If we continued to add the kanamycin concentration to 100μg/ml, the value of k lessened slowly. Our experiments showed that the value of k in the absence of kanamycin was always greater than those in the presence of kanamycin. So, kanamycin had inhibitory effects on the growth of E. coli B1. The higher the kanamycin concentration was, the smaller the value of k was. We established the equation for lnk-C and calculated the IC₅₀ which is about 88.49μg /ml.A microcalorimetric technique was used to evaluate the influence of Mg²⁺on the growth of E. coli B1. We obtained the thermogenic curves of E. coli B1 growth at different Mg²⁺ doses. In order to analyze the results, the growth rate constants k, the maximum power P_m, and the heat effects Q were determined, which showed that values of P_m and k were all first increased and then reduced with the concentrations of Mg²⁺ increasing from 0 to 4.4mg/ml. However within the same ranges, the time (t_D) of reaching the maximum effect in the log phase and the time (t_S) of maintaining the maximum effect in the stationary period were first decreased and then increased. During the growth of E. coli B1 in the presence of Mg²⁺ ion, the heat given out was greater than that without Mg²⁺ ion. So, Mg²⁺ ion has a promotive action on its growth when Mg²⁺ ion varied in the concentration ranges of 0-2.2 mg/ml. When Mg²⁺ concentration reached 2.2mg/ml, P_m attained the extremum, 53.1μW. With more Mg²⁺ ion (>2.2 mg/ml) added, the promotive effect would decrease intensively. If Mg²⁺ ion varied from 2.75 mg/ml to 4.4 mg/ml, the value of k was independent on Mg²⁺ concentration and maintained a fixed value. Therefore, Mg²⁺ ion has a promotive action on its growth. On the basis of the relationship between k and C (Mg²⁺), we calculated the optimal Mg²⁺ dose, which is 2.09 mg/ml, and the optimal value of k at this dose, which is 0.0384mm^-1.The effects of different original cell concentrations on the growth of E. coli B1 were studied by microcalorimetry. We got more information, not only thermal data but also kinetic data, and the corresponding thermokinetic equation. Our results showed that a special effect caused by cell concentrations existed during the growth of E. coli B1. Just because of the effect, with original cell concentrations increasing, the maximum thermal power added, the thermal power produced by one cell decreased, and the total heat effects maintained. We can see that with original cell concentrations increasing, the rate constant k and the maximum thermal power P_m enhanced gradually, suggesting that high original cell concentration promoted the growth of E. coli B1. When original cell concentration varied from 0.39×10⁷to 1.79×10⁷ cell/mL, the values of k, P_m and Q_log increased from 0.0281 min^-1, 40.1μW, 126.62 mJ to 0.0425 min^-1, 62.8μW, 473.981mJ. At the same time, the values of Q_sta decreased from 372.93 mJ to 30.144 mJ, and the total heat effects Q kept a fixed value, 500 mJ. In all, the addition of original cell at high concentrations may promote the growth of E. coli B1.According to our experimental results, we found that the influences of different pH on the growth of E. coli B1. With pH increasing from 6.06 to 6.93, the values of k and P_m increased from 0.0207 min^-1, 33.0μW to 0.0394 min^-1, 59.6μW. The growth of E. coli B1 was promoted. If we continued to enhance the value of pH to 8.19, the values of k and P_m will decreased gradually from 0.0394 min^-1, 59.6μW to 0.0167 min^-1, 26.1μW. The growth of E. coli B1 was inhibited .On basis of the equation for k - pH, we calculated the optimal pH, which is 6.74, and the optimal value of k at this pH, which is 0.0407min^-1.