| trans-4-Hydroxyproline is widely applied in many fields, such as the pharmaceutical, chemical, cosmetic products and food industries. trans-4-Hydroxyproline was mainly obtained through the hydrolysis of animal collagen originally, but the yield was very low. Proline 4-hydroxylase found from Dactylosporangium sp. RH1 could transform free L- proline to trans-4-hydroxyproline. In this thesis, we knocked out the sucA gene of E. coli and introduced the plasmid pUHVT4 containing trans-4-hydroxylase gene（P4H） into the sucA-knockout recombinant E. coli. Fermentation optimization was then conducted at shake flask level to improve conversion efficiency of L-proline.The Red homologous recombination system was used to knock out sucA gene of E. coli BL21（DE3）, E. coli BL21（DE3）ΔputA and E. coli W3110ΔputA. At the same time, gene P4 H was inserted on the chromosome. Introducing plasmid pUHT4 and pUHVT4 into E. coli BL21（DE3）ΔputAΔsucA, after having a fermentation, the highest production of trans-4-hydroxyproline of E. coli BL21（DE3）ΔputAΔsucA/pUHVT4 reached 64.32 mg·L-1, which was 1.1 times as high as E. coli BL21（DE3）ΔputAΔsucA/pUHT4. This demonstrated gene vgb from plasmid pUHVT4 could effectively promote the synthesis of trans-4-hydroxyproline.Importing plasmid pUHVT4 into the sucA-knockout recombinant E. coli strains, E. coli BL21（DE3）ΔputAΔsucA/pUHVT4 was found having the strongest capacity of producing trans-4-hydroxyproline after a fermentation. By comparison, E. coli BL21（DE3） was found more suitable for producting trans-4-hydroxyproline than E. coli W3110. Among all strains, E. coli BL21（DE3）ΔputAΔsucA/pUHVT4 had the highest whole-cell enzyme activity, which reached 4.26 U·mg-1.Using E. coli BL21（DE3）ΔputAΔsucA/pUHVT4 as experimental strain, after single factor, orthogonal experiment and further optimization, the optimal medium components were obtained: glucose 4 g·L-1, glycerol 10 g·L-1, K2HPO4 11 g·L-1,（NH4）2SO4 1 g·L-1, corn steep liquor 16 g·L-1, NaCl 0.75 g·L-1, MgSO4 0.3 g·L-1, CaCl2 0.005 g·L-1 and FeSO4 1 mmol·L-1. The most suitable inoculation amount and initial pH were 6%(v·v-1) and 8.5, respectively. After a 12 h fermentation, the production of trans-4-hydroxyproline reached 1.57 g·L-1, which was about 5.6 times as much as initial. Then in a 5 L fermenter, expanding culture was conducted. The results showed: better carbohydrate supplement rate and the ratio of initial glucose and corn steep liquor content were 1.8 g·L-1·h-1 and 1:1, respectively, and the production of trans-4-hydroxyproline had a enhancement, which reached 41.5 g·L-1 at 64 h. |
PDF Full Text Request