| Because Penicillin acylase is capable of catalyzing β-Mactam nucleus and D-amino acid analogues to compound new β-lactam antibiotics under acidic conditions, so it is an important enzyme for pharmaceutical industry. Synthesis of β-lactam antibiotics in the traditionally chemical method not only has the shortcomings of high reaction conditions, multi-steps reaction, high cost and low efficiency, but also needs to use the highly toxic reagents, such as PCl5, pyridine, NOCl, polluting the environment extremely easily. In contrast, enzymatic synthesis method has the advantage of simple production process, less environmental pollution, mild reaction conditions, the absolute specificity and lower production costs. Therefore, it is of great practical significance to produce penicillin acylase by microbial fermentation.This paper used recombinant Escherichia coli as the experimental strain to study the optimization fermentation conditions of penicillin acylase, batch fermentation kinetics, immobilization of penicillin acylase and enzymatic synthesis of amoxicillin. The experimental results were as followed:Optimization of the fermentation conditions of penicillin acylase:Single factor experiment was used to determine that the optimal concentration range of peptone, glycerol, and IPTG were respectively 15～25 g/L,15～20 g/L, and 50 μg/mL, and the optimal fermentation temperature and pH were respectively 28～32℃, pH6.5-7.5. Box-Behnken design was used to optimize the fermentation conditions of synthesis of penicillin acylase by recombinant Escherichia coli, and statistical analysis software SAS 8.1 was used to analyze the experimental data, the optimum fermentation condition obtained was that the optimal concentration of peptone and glycerol were respectively 23 g/L,23 g/L, the optimum fermentation pH was pH7.2. The average enzyme activity of penicillin acylase was 3 989.46 U/L in the optimum fermentation conditions,6.5% higher than that of the single factor experiment.Batch fermentation kinetics:The fermentation synthesis of penicillin acylase by recombinant Escherichia coli was amplificated in the 5 L fermentor, according to the Luedeking-piret equation and logistic equation, and the non linear fitting was applied to the fermentation experiment data of synthesis of penicillin acylase by recombinant Escherichia coli using origin 8.0 software, and the bacteria growth kinetics model, synthesis of penicillin acylase kinetics model and substrate consumption kinetics model of batch fermentation synthesis of penicillin acylase were established, and they were respectively shown below:Cell growth kinetics model:Synthesis of penicillin acylase kinetic model:Substrate consumption kinetics model:The model could well simulate the dynamic process of batch fermentation synthesis of penicillin acylase by recombinant Escherichia coli, providing a reference basis for the industrial production of penicillin acylase.The immobilization of penicillin acylase:Single factor experiment was used to determine the optimal range of the factors having effect on the immobilization of penicillin acylase, and the optimal range was optimized by Box-Behnken design. The results showed that the optimum immobilization conditions were:temperature 26℃, pH8.2,31 h, carrier dosage 0.11 g/mL. Average enzyme activity of the immobilized enzyme and the recovery rate of enzyme activity were respectively 130.84 U/g, 60.72% in the optimum immobilization conditions.Enzymatic synthesis of cefadroxil:Temperature, pH, side chain and the parent nucleus molar ratio, investment amount of enzyme and the parent nucleus concentration of enzymatic synthesis of cefadroxil were optimized. Experimental results showed that under the condition of 20℃, pH6.5, molar ratio 1.2:1, enzyme amount 20 g, parent nucleus concentration of 13% of the technological, the reaction conversion rate reached 99.47%. Conversion reaction was repeated 100 batches under the optimum conditions, and the average conversion rate high reached 98.32%. |
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