High Efficient Production Of Purine Nucleoside Phosphorylase And L-tryptophan With Recombinant Escherichia Coli

Purine nucleoside phosphorylase (PNPase, E.C.2.4.2.1) is a key enzyme in the purine salvage metabolic pathway. It could catalyze the synthesis of nucleosides, and has great potential in anti-tumour gene therapy. On the other hand, L-tryptophan (L-trp) is one of the eight essential amino-acids for human and widely applied in the fields of medicine, food additives and animal feeds. This study has respectively implemented the high production of PNPase and L-trp in the recombinant Escherichia coli.Firstly, the expression vector of PNPase was constructed and transformed into E. coli BL21 (DE3) cells to form the recombinant strain. After the optimization of induction and expression, soluble and functional PNPase was effectively expressed, with the maximum yield of 196 U/mL. Moreover, the investigation of substrate specificity indicated that the recombinant PNPase was almost the same as the non-recombinant PNPase. Meanwhile, the immobilization of recombinant E. coli cells was also carried out. Agar was found to be a suitable matrix for whole cell entrapment. The followed enzymological characterization, kinetic analysis and stability comparison had suggested that the immobilization process reduced the substrate affinity and catalytic efficiency of recombinant cells, but significantly enhanced the stability and reusability of these cells. The reaction catalyzed by immobilized cells only complied with the Michaelis-Menten equation at high substrate concentration, the evident deviation was observed at low substrate concentration due to the diffusion limitation; while the reaction catalyzed by free cells completely obeyed the model under both conditions. Further more, the immobilized whole cell biocatalyst was applied to synthesize ribavirin as the model reaction. The reaction parameters were first optimized in the free cell catalyzed rabavirin synthesis, giving the highest yield of 80.8%. When rabavirin was synthesized by immobilized cells, the yield slightly decreased to 70.2%, but the reusability was enhanced. When 18.8 g/L immobilized cells were incubated with substrates in 10 continuous round reactions for 4 hours, the measured yield exhibited no obvious decrease.Secondly, the high production of L-trp via the fermentation of recombinant E. coli was systematically studied. The seed and fermentation media, as well as the culturing conditions were optimized. Batch and fed-batch fermentation were also carried out in the lab-scale fermentator. The results indicated that the addition of phosphate significantly affected the production of L-trp in the batch process, and the optimal initial concentration of phosphate was determined as 20 g/L in the fed-batch process. Moreover, the exponential feeding of glucose could obviously enhance the performance of fermentation, the maximum titer reached 10.6 g/L and the productivity was 0.45 g/(L-h). Further improvement of feeding strategy based on the substrate-feedback was able to maintain the low concentration of glucose in the broth, which greatly increased the titer of L-trp to 25.5 g/L. Based on these results, a new exponential feeding method was established for L-trp production, with the titer of 24.9 g/L and the average productivity of 0.58 g/(L-h).Furthermore, the effects of additives on the fermentation production of L-trp were also examined. The results demonstrated that non-ionic surfactants could effectively promote the accumulation of L-trp, but antibiotics and cation surfactants had no obvious effect. Thus, Tween 60 and PL61 were supplemented into fed-batch fermentation, in which the titer of L-trp was increased to 32.3 and 35.5 g/L, respectively. Based on the principle of metabolic engineering, the metabolic fluxes in the biosynthetic pathway of L-trp were estimated. Results showed that Tween 60 and PL61 could both change the flux distribution, leading to the increased flux of L-trp biosynthesis. In addition, the separation and purification of L-trp from fermentation broth via strong-acid cation exchange resin was developed. An adsorption empirical model was established, related adsorption kinetic and thermodynamic analyses were also carried out. With the highly effective bed-column separation process and other procedure optimization, the total recovery of L-trp from fermentation was 68.8%, and the purity of L-trp could reach 97.5%.In conclusion, two important fermentative products were efficiently produced in different recombinant E. coli strains, respectively; and these bioprocesses were systematically investigated and optimized. The results from this study provided valuable data and basic for the industrial production of PNPase and L-trp.