Breeding Of Brevundimonas Diminuta For L-proline And Optimization Of Fermentation

L-proline is the only imino acid among all20amino acids involving in proteincomposition, which is one of the most important amino acids for compound infusion in themedicine field. It has good chiral catalytic activity in chemical synthesis as small moleculecatalysts and has a special biological function in resisting the extreme environment forL-proline in vivo.Moreover, it plays an important role in maintaining normal life activities ofthe organism.With the wide application of L-proline in medicine, food, chemical industry andagriculture, its demands are constantly increasing.This research mainly focused on a strain producing L-proline, which was preserved inour laboratory. By determining the characterics of physiology biochemistry as well as analysisof16S rDNA gene sequence of JNPP^-1, the JNPP^-1was identified to be a strain ofBrevundimonas diminuta in the status of taxonomy. We obtained a mutant strain which couldproduce L-proline with a high level by ultraviolet （UV） andN-methyl-N-nitro-N-nitrosoguanidine （NTG） treatment. And then the optimization offermentation condition was performed. Finally, the fed-batch fermentation was studied in5Lfermenter. The results are as follows:1． Through the characteristics of morphology, physiology biochemistry and analysis of16SrDNA gene sequence of JNPP^-1, the strain of JNPP^-1was proved to be a strain of B.diminuta.At present, there was no report about the B. diminuta producing L-proline in literature in theworldwide.2． B. diminuta JNPP^-1was used as the original strain for stepwise mutation by ultraviolet（UV） and N-methyl-N-nitro-N-nitrosoguanidine （NTG） treatment. A stable and L-prolineover-producing mutant JNPP-NSS （SGr、Sucg、NaClr） was obtained. At the end offermentation, the maximum concentration of L-proline reached45g·L^-1, which was42.0%higher than that of the original strain.3． The effect of the medium composition on L-proline production in flask was studied bysingle-factor experiment and orthogonal experiment design.The results showed that theaddition of glucose and sodium glutamate could make a significant effect on L-proline yield.The optimal medium compositions were determined as (g·L^-1): Glucose100,（NH4）2SO455,Yeast extract30, K2HPO42.5, MgSO4·7H2O0.3, ZnSO40.01, MnSO40.01, Sodium glutamate50, CaCO34.5, VB150μg·L^-1, Biotin50μg·L^-1,30mL/500mL, pH8.0. After62h ofincubation, the mutant strain could accumulate49.2g·L^-1of L-proline, which was9.3%higher than that of the original culture condition.4． The experimental results indicated that lag phase was long, high yield or productivity ofL-proline could not be achieved in a batch process. A fed-batch culture was proposed byfeeding of glucose and sodium glutamate to improve the L-proline production. Theexperimental results showed that the yield and productivity of L-proline reached57.6g·L^-1and0.944g·L^-1·h^-1at the feeding rate of0.9g·L^-1·h^-1sodium glutamate, which were18.2%and20.9%higher than that of shaking flask fermentation, with the initial sodium glutamate concentration of50g·L^-1.