High Density Fermentation Of Recombinant Pichia Pastoris Expressing PGA And Immobilization Of The Enzyme

Penicillin G acylase is an important enzyme preparation which is used for antibiotic intermediate6-aminocephalosporanic acid (6-APA) and7-amino deacetylation of cephalosporin acid (7-ADCA) production. Penicillin G acylase is also available to catalyze condensation reaction of D-amino acid analogs and β-lactam nucleus under acidic conditions, the reaction generates a new β-lactam antibiotics. A large amount of production and application of penicillin G acylase attach great importance to the widespread uses and lower prices of penicillin and cephalosporin antibiotics. The advantages of enzymatic synthesis method were the absolute specificity of the reaction, avoiding environmental pollution, providing mild reaction conditions and reducing production costs. Therefore, the use of microbial fermentation expressing penicillin G acylase has a positive and practical significance on completing the fermentation processes. The main results can be listed as follows:Optimization of fermentation conditions:To study the optimization of fermentation conditions of recombinant Pichia pastoris expressing penicillin G acylase, by using single factor experiments, the optimum ranges were determined:optimum glycerol concentration40g/L, ammonia concentration0.1%, methanol concentration1.0%, pH5.5, inoculum10%. Then we optimized the fermentation conditions of Pichia pastoris expressing penicillin G acylase with the Box-Behnken experimental design. The model showed R2=0.9813by means of response surface analysis, indicated the quadratic regression equation was good. After the optimization, the enzyme activity reached8680±12U/LThe fermentation kinetics modeling:Scale-up studies of Pichia pastoris fermentation expressing penicillin G acylase were carried out in a5L fermenter. The activity of penicillin G acylase in fermentation broth reached46345U/L. According to the data showed in the growth and product formation of the strains at different stages of dynamics and fermentation experiments, we analyzed the progress on the basis of the application of Matlab to fit the curve, then we established the cell growth models, the synthesis of penicillin G acylase models and substrate consumption dynamics models. These models are able to describe the dynamic processes of Pichia pastoris batch fermentation expressing penicillin G acylase, they heve provided a reference for further subsequent amplification.Purification:In turns, we used ultrafiltration concentrated, ammonium sulfate purification and ultrafiltration desalination to purify the enzyme solution of penicillin G acylase which derived from Pichia pastoris fermentation. Then the highly purified penicillin G acylase was obtained. After purification, the activity of penicillin G acylase enzyme solution reached31.3U/mg, the purification was increased6.38times, the recovery was49.78%.Penicillin G acylase immobilized:Referring to the literature and experimental comparisons to decide which immobilized carrier we could use. Then we studied the conditions of immobilizing penicillin G acylase process with LX-1000EP-2resin. Single factor tests and Box-Behnken Design were used to analyze the optimal immobilization conditions:pH8.1, temperature25℃, the amount of carrier0.8g, immobilization time24h. The enzyme activity of the immobilized penicillin G acylase was356.89U/g, the enzyme activity recovery rate was59.82%.