Breeding Of Pseudomonas By Mutagenesis And Optimizing Of Fermentation Conditions

D-Phenylglycine, which can be used to produce β-lactam antibiotics and cephalosporin like amoxicillin etc. is a kind of most important precursor. It is usually produced through three ways, but the most economic way is producing by bio-enzyme. Pseudomonas. putida ,which is researched in this dissertation, can excrete two enzymes at the same time: D-hydantoinase and D-carbamoylase. These two enzymes can catalyse D-hydrocyphenylhydantoin to D-Phenylglycine directly.the wild Pseudomonas . putida produces low enzyme activity, the study mainly induced the wild strains to produce high enzyme activity.The indexes of starting bacteria was validated. Through the comparison of definition, the growth curve was make out. And the results showed: the best time of mutagenesis was after 24h culture of bacteria. The orderness between enzyme activity and growth curve was anyalysed by PDAB, which showed that the enzyme activity would be higher at the end of the logarithm time. And the enzyme activity of starting bacteria was 0.107μ/mL which was measured by HPLC.In this study, UV, MW, US and their combination were used to mutagenize the wild strains, then the new bacteria were screened out with substrate analogue. Finally, several new bacteria were screened out which excrete more D-hydantoinase and D-carbamoylase. Among those new bacteria, the total enzyme activity of FUMS06 can reach 0.440μ/mL, which enhanced 311.21 % compared with the starting bacteria.The culture medium and the fermentation conditions were optimized based on the former culture medium and formal fermatetion conditions. The results showed mat the enzyme activity of FUMS06 enhanced more. And the best composition was as following: corn steep liquor 3.0%,glucose 2.5%, revulsent 3.5%,NaCl 0.3%.The FUMS06 grew well on the optimized medium, compared with the formal medium The enzyme activity enhanced 11.8%. The best fermentation condition was as following: The inoculum amount 10%,loading amount 40mL,culture time 26h,initial pH7.4.The finally result of fermentation was idealic, and the activity of FUMS06 can remain after several generation breed, and the highest enzyme activity was 0.492μ/mL,which enhanced 359.80% compared with the starting bacteria .