Optimization Of Penicillin G Acylase Production By Recombinant Bacillus Subtilis Using Response Surface Analysis

Penicillin G Acylase is the key biological catalyst in the industry production of β-lactam antibiotics, and it is very important in the synthetic antibiotics industry.Bacillus subtilis expression system is a kind of efficient microbial expression system which has obvious advantages over E.coli expression system. Bacillus subtilis expression system can secrete the target proteins into the extracellular which can be easily separated and purified. Response Surface Methods and Designs were used to optimize the fermentation conditions for Penicillin G acylase production by Recombination B.S168. Initial screening by Plackett-Burman Methods and Designs revealed that the fermentation temperature and yeast extract were the significant factors among19factors. Further optimization with Central composite Design and Response surface analysis predicted that the final enzyme activity can reach28.2U/mL when the temperature was34.1℃and yeast extract was8.81g/L. And the shake flask experiments were also used to verify the results which showed the predict value and the actual experimental data fitting can reach99%. The enzyme activity of optimization conditions was as2.19times as the initial conditions. In accordance with the optimization conditions, we used the15L fermentor to expand fermenting, and studied the influence of stream feeding to the production of PGA. Eventually, the highest PGA activity of the fermentation liquid could reach60U/mL at40hours when we stream feeding in bacterial medium logarithmic growth period which also reached the leading domestic level.