Process Optimization Of Efficient Biotransformation Of L-Phenylalanine To Phenyllactic Acid

Phenyllactic acid?PLA?is a safe,non-toxic,natural and broad-spectrum antibacterial substance that can be used in industries such as medicine,food,cosmetics and agriculture.The recombinant Escherichia coli BL21?DE3?harboring pRSF-aad-ldh₁₀-fdh was used in this study to reduce the cost of fermentation medium and increase the yield of PLA in the 3 L fermenter.The concentration and type of carbon source and nitrogen source in the fermentation medium were optimized at the shake flask level and the culture conditions of the bacteria in the 3 L fermenter were optimized.Then the effects of rotational speed and aeration on the dissolved oxygen value?DO?and cell density were investigated to lay the foundation for the fed-batch culture.In addition,the induction conditions of the co-expressed enzymes in the 3 L fermenter were optimized.The inducer concentration,induction timing and induction temperature were determined by investigating the ability of whole cells to convert L-phenylalanine?L-Phe?into phenyllactic acid.In the whole cell biotransformation process,adding glucose could significantly increase the productivity.Therefore,this paper also discussed the mechanism of glucose in increasing the productivity of phenyllactic acid from L-Phe.The main results in this study were described as follows:?1?The optimal fermentation medium composition:glucose 4 g/L,FM802 Angel Yeast extract powder 24 g/L,KH₂PO₄ 2.31 g/L,K₂HPO₄ 12.54 g/L.The culture condition of E.coli BL21?DE3?harboring pRSF-aad-ldh₁₀-fdh at the shake flask level:liquid volume was 25 mL in 250 mL flask and the inoculum size was 4%,which was carried out at 37?,pH 7.0 and220 r/min for 14 h.?2?The rotational speed,aeration and fed batch feeding strategies?constant speed flow,pH-stat and DO-stat?were optimized.The optimal feeding strategy was to feed the 400 g/L glucose at a constant rate of 10 mL/h at 400 r/min and 1.5 vvm for 10 h after the cells were cultivated for 3 h.Under the optimal conditions,the dry cell weight were 13.71 g/L.?3?The induction conditions in the 3 L fermenter were optimized.The optimal inducer concentration was 0.08 mmol/L;the induction timing was 2 h and induction temperature was25°C.?4?The whole cell transformation conditions in the 3 L fermenter were investigated,including NADH supply?ammonium formate and glucose?,substrate concentration,temperature,pH,rotation speed and aeration.The results showed that the glucose was used as a source of coenzyme NADH regeneration.When the dry cell weight was 30 g/L,the 30 g/L substrate L-Phe was converted into phenyllactic acid at 37°C,pH 7.0,300 r/min and 1.0 vvm in the 3 L fermenter,with a yield of 25.4 g/L and a conversion rate of 84.7%.?5?The changes of intracellular ATP levels,the changes of DCW,the yield of phenyllactic acid from glucose and the integrity of cell membrane were investigated to figure out the mechanism of glucose in increasing the productivity of phenyllactic acid from L-Phe.The results showed that adding glucose could increase the intracellular ATP levels,alleviate ATP reduction caused by phenyllactic acid and provide ATP to maintain cell activity during the whole cell biotransformation;before and after the whole cell transformation process,adding glucose had little effect on DCW and the integrity of cell membrane;E.coli BL21?DE3?harboring pRSF-aad-ldh₁₀-fdh could directly synthesize phenyllactic acid from glucose and the highest conversion rate was 0.433%.