Regulation On Fermentation Process Of Producing PGA With A Recombinant Bacillus Megaterium System And Immobilization Of The Enzyme

PGA is widely used to catalyze and pyrolyze penicillin G and spore mildew element G to produce6-amino blue mildew alkane acid and7-aminos-3-escapes the acetoxy spore alkane acid, as an important medicine industrial enzyme. PGA also can catalyze the reversed reaction, in other words, under the acidic condition, catalyzes the D-amino acid synonym the condensation to produce the new Beta-lactan antibiotic with Beta-the lactan parent nucleus (for example6-APA,7-ADCA). It has many bad effects to produce6-APA by chemical means, such as multiple steps, low yield, hard reaction conditions and high cost. Further, it needs highly toxic reagents such as pyridine, PCl5, N0Cl, which are prone to pollute enviroments. Compared with chemical methods, enzymatic synthesis owns absolute specificity and moderate reaction conditions while can avoid enviroment pollution. In addition, the cost reduced by more than10%, so the production process will be economical to combine6-APA synthesis using enzymatic with production of penicillin by fermentation. Then it has realitic significance to produce PGA by fermentation and rise productivity and gain complete rfermentation technology.Our test strain is Bacillus megaterium through genetic modification. This paper is concerned with optimization of fermentation conditions, kinetic model of batch fermentation, isolation and purification and immobilization and the characterization of PGA, and the main results are as followed:To optimize the fermentation conditions, monofactorial experiment and response surface methodology were combined. After monofactorial experiment, the optimal carbon source, nitrogen source and their best concentration and fermentation time and temperature could be determined as followed:25-35g/L、10-20g/L、36-44h、29-37℃. Box-Behnken and response surface methodology were designed for the optimization:glucose32g/L, casein17g/L, time42.5h, temperature34℃and in this case, enzyme activity of PGA was33514.28U/L which increased by8.66%than before.The fermentation was magnified in fermenter of5L and in this optimal condition the activity was39887U/L. In the condition of Matlab, the kinetic model of cell growth, the kinetic model of synthesis of PGA, the kinetic model of substrate consumption were obtained by genetic algorithm. The result is as followed:(1) The kinetic model of cell growth (2) The kinetic model of synthesis of PGA(3) The kinetic model of substrate consumptionThis model described dynamic process of batch fermentation of recombinant Bacillus megaterium well, which can provide references for latter scale experiment.To study the isolation and purification of Penicillin G Acylase, ultrafiltration ammonium sulfate salting, concentration were designed. And the result was that the activity after purification rose29.16U/mg, purification times was1.59, and the specific activity was60.59%.The process conditions of immobilization PGA with epoxy were explored. Box-Behnken and response surface methodology were designed for the optimization:pH8.1, temperature29℃,carrier usagelg, time24h, and in this case, enzyme activity was363.76U/L and enzyme recovery was62.82%. After a series of determination of properties, the results suggests the optimal pH was9.0and optimal temperature was60℃, what was more, the continuous operation stability of the immobilized enzyme was well, which shows it’s viable to use epoxy to immobilize PGA.