Study On The Fermentation Process Of The Genetically Engineered Escherichia Coli Bacteria Strain For The Biosynthesis Of Theanine

This paper studied the optimization of cultivation medium and fermentation condition in flask, the stability of the recombinant plasmid pET32a-GGT, biosynthesis of theanine by immobilized cells, scale-up fermentation process in 30L fermentor of the genetically engineered Escherichia coli strain ofγ-Gltamyltranspeptidase for the biosynthesis of theanine.The main results are as follows:1,Plackett-Burman design and Response Surface Methodology(RSM) was used in this study, the optimum cultivation medium were glucose 10.39g/L, yeast extract 6.88g/L, K2HPO4 7.10g/L, tryptone 10g/L, NaCl 10g/L, MgSO4·7H2O 1g/L, the optimum fermentation condition were: initial pH7.32, cultivation time 6.67 h, IPTG induction temperature 31.51℃, medium loadage 50mL/250mL, inoculum percentage 1%, IPTG induction time 4h. The experimentalγ-GGT activity obtained was 4.64U/mL. The yield of theanine from L-Gln and ethylamine was 35.18g/L, conversion rate of L-Gln was 57.7%.2,The recombinant plasmid didn't appear obvious gene deletion, showed structural stability after 100 generations. However, the plasmid was showed segregational instability, the plasmid-free cells appeared after 20 generations, and 18% cells was plasmid-harboring cells after 100 generation under no Amp selection pressure. It was structural and segregational stability under optimizing culture medium and condition during the fermentation process.3,The cells of the genetically engineered Escherichia coli were entrapped by sodium alginate solution (20g/L) and were treated by 0.05% glutaraldehyde after immobilization. The activity yield of the immobilized cells was 79.2%. The storage stability and the operational stability were greatly improved after immobilization. Continuous theanine production was carried out in a Packer Bed Reactor (PBR) ,the average yield of theanine was 25.40 g/L, and the average productivity was 6.35g/L/h。4,The fermentation procees in 30L fermentor were: 30L fermentor containing 12L optimum cultivation, inoculum percentage 5%, supplementing with 5mL acid butyl phosphoric as foam killer, shaking at 250r/min, cultivation temperature 37℃, adding 100ml glucose(10g/L) when Dissolved Oxygen(DO) under 40%, controlling fermentation pH at 7.3 with 5mol/LNaOH, adding 0.05mmol/L IPTG after 6h fermentation,induction temperature 31.5℃for 4h.The OD600 was 19.2,γ-GGT activity was 1.22U/ml. The yield of theanine was 8.06g/L, and conversion rate of L-Gln was 13.22%.