Study On The Fermentation Engineering Of E.coli To Produce Recombinant Hyperthermophilic Esterase

Hyperthermophiles represented by bacterial and archaeal species grow optimally at temperatures range from 80 °C to 110°C. Enzymes from these organisms developed unique structure-function properties of high thermostability and optimal activity at temperatures above 70°C. Aeropyrum pernix K1 has been isolated in 1993 from coastal solfotaric thermal vent. It grows in the range of 90–98°C with an optimal temperature of 95 °C and its genome sequence has been solved. We cloned the gene APE1547 and transformed the recombinant plasmid into E. coli strain to express hyperthermophilic esterase. The hyperthermophilic esterase has the potential uses in industrial processes, i. e., organic synthesis reactions. Bioprocess development for the production of hyperthermophilic esterase is important from an industrial point of view. In the flask experiments, it was confirmed that the recombinant hyperthermophilic esterase got the best production at 37℃, pH7.8 , 2% inoculation and round speed 180 rpm. When the OD600 reached 0.8, the induction was performed. After that the culture continued for another 12 hours to get the best production. The plasmid stability is usually sustained by the ampicillin pressure. But as ampicillin is too expensive for large scale culture of recombinant E.coli, we substitute penicillin sodium for ampicillin to sustain the plasmid stability. The optimal amount of the penicillin sodium concentration is 6μg/ml. The lac promoter and its derivatives are widely employed for the purpose of controlled expression systems and in most cases the gratuitous inducer IPTG is used for foreign gene expression, in spite of its higher cost and toxicity towards humans when compared to lactose. Lactose serves simultaneously as inducer and as carbon and energy source. In this experiment, we confirmed the optimal inducer lactose concentration in flask, which is 0.5mM. The optimization of nutrient levels for the production of recombinant hyperthermophilic esterase by E.coli was carried out using response surface methodology (RSM) based on the central composite rotatable design (CCRD). Response surface methodology is an empirical statistical modeling technique employed for multiple regression analysis using quantitative data obtained from properly designed experiments to solve multivariable equations simultaneously. RSM is used to determine the optimum response of the yield of under a range of culture conditions. The significant independent variables of the medium components are evaluated. Regression analysis was performed on the data obtained from the design experiments. A 24 central composite rotatable design was used to study the combined effect of the nutritional constituents like yeast extract, peptone, mineral salt and trace metals. The P-value of the coefficient for linear effect of peptone concentration was found to be 0.0081, and for trace metals solution the P-value was less than 0.0001, suggesting that these were the principal variables having the significant effect on the hyperthermophilic esterase production. The final composition of the medium is as follows: peptone 15.4g/L, yeast extract 8.7g/L, mineral salt solution 21.8ml/L and trace elements solution 0.5ml/L. This medium produces theoretically hyperthermophilic esterase activity 269.17 U/ml and in practice 284.58 U/ml, which is a considerableimprovement as compared to the results of previous studies. Recombinant hyperthermophilic esterase was produced from the agricultural waste product corn steep liquor. The effect of corn steep liquor, mineral salt and trace metals on hyperthermophilic esterase production was investigated by means of a five-level three-factor central composite rotatable design. The final composition of the medium is as follows: corn steep liquor (24.3g/L), mineral salt solution (16.5ml/L) and trace elements solution (12.7ml/L). The predicted response was 261.60U/ml and the actual response was 251.39U/ml, which proves the validity of the culculation. The recombinant enzyme should be produced in a cost-effective way because the pharmaceutical companies prefer the cost-effictive media. In this experiment, the efficiency of corn steep liquor in cultivating recombinant E.coli was tested. From the results, it is shown that corn steep liquor is an efficient complex medium for recombinant enzyme production, compared with costly traditional medium such as yeast extract and peptone. Because of its low cost and high efficiency, the corn steep liquor will be the primary substrate for the scale-up production of recombinant enzyme, at least, for the hyperthermophilic esterase. Acetate is produced when E.coli is grown under anaerobic or oxygen-limiting conditions; however, E.coli cultures growing in the presence of excess glucose can also produce acetate even under aerobic conditions. Acetate is produced when carbon flux into the central metabolic pathway exceeds the biosynthetic demands and the capacity for energy generation within the cell. Saturation of the tricarboxylic acid cycle and the electron transport chain may be the main cause. A high concentration of acetate reduces growth rate, biomass yield and maximum attainable cell densities. Furthermore acetate has been reported to have a greater detrimental effect on recombinant cells than on non-recombinant cells, and recombinant-proteinproduction is significantly reduced by acetate accumulation. A number of strategies have been developed to reduce acetate formation in fed-batch culture by controlling the specific growth rate by limiting essential nutrients such as sources of carbon or nitrogen. The simple feeding strategies such as constant-rate, stepwise increase of the feeding rate and exponential feeding have been used to obtain high cell-densities of E.coli in fed-batch cultures. The exponential-feeding method has been developed to allow cells to grow at constant specific growth rates, it also provides the advantage to minimize the acetate production by controlling the specific growth rate below the critical value of acetate formation. The feeding rate that allows exponential growth with constant specific growth rate can be calculated using the equations, which is derived from a simple mass balance with the assumption of constant cell yield on substrate. In this study, a series of bioreactor cultivations with similar growth phases was performed to develop the subsequent induction phase with the aim of achieving high levels of hyperthermophilic esterase expression using lactose as the inducer molecule. 10L corn steep liquor medium with the 10g/L glucose was used for the batch fermentation. The transition from the batch to the fed-batch phase is indicated experimentally by the rapid raise in DO concentration, which corresponds to the depletion of the glucose added at the beginning of the batch phase. During the fed-batch phase, the 500g/L glucose was added and the specific growth rate was kept at 0.15h-1. When the OD600 gets 100, the induction was performed with lactose. Two variables were considered to be the most important ones in the development of adequate induction strategies: the specific amount of lactose added and the way in which lactose is transferred to the reactor. In run A, the total specific amount of lactose added was 1.4g per gram of dry cell weight and the interval was 1 hour. The resulting hyperthermophilic esterase activity achieved in this run was considered to be a low one. Therefore, the specificamount of lactose, which was threefold, reaching 4.3g/g to be added in the next run, which was performed in three pulses and the interval was 1 hour too. The hyperthermophilic esterase activity achieved through this strategy was 190U/ml, which is very low because of the DO limitation. In order to increase the production, the specific amount was kept at 4.3g/g, but the interval is 2 hours, with the aim of avoiding inhibitory effects that could eventually occur due to a high residual concentration of lactose in the medium. The hyperthermophilic esterase activity attained 800U/ml through this new strategy. It was considerably higher than the ones attained before. Another attempt was made to improve hyperthermophilic esterase by introducing a rich nutrient source (corn steep liquor) together with the second and third lactose pulses, which might add new nutrient to produce heterologous protein. The hyperthermophilic esterase activity got 1100U/ml, and it was adopted as the final mode to produce the hyperthermophilic esterase. This work is the first reported study of the E.coli disruption by the thermolysis method to release the recombinant thermostable enzyme. The membrane structure of E.coli became permeable by heat treatment, which results in the hyperthermophilic esterase release. At the same time of disruption, the hyperthermophilic esterase was purified because most of the intracellular motley proteins would be denatured and precipitated by heat treatment. The effects that affect the disruption efficiency have been investigated over a range of operating conditions. The degree of disruption was found to be highly dependent on both the incubation temperature and the buffer system. It reached maximum when the cells are heated in the pH7.0 Tris buffer at 80℃. In addition, the disruption efficiency is also dependent on the cell concentration though the dependent is minor in the range tested. The cell disruption process can be modeled by a first order kinetic. After culturedin medium containing corn steep liquor, the cells were disrupted in the fermentation broth in situ. Furthermore, the effect of thermolysis and chemical method was compared. A series of hyperthermophilic esterase samples with different purities were obtained. In this study, we found a suitable way to produce the recombinant hyperthermophilic esterase from small scale to pilot scale and the fermentation process was optimized. It provides a base to the industrial scale production of hyperthermophilic esterase.