Studies On Biosynthesis Of Phenyllactic Acid By Lactobacillus Sp.SK007

Phenyllactic Acid(PLA), also calledβ-Phenyllactic acid or 3-Phenyllactic acid. It can inhibit many kinds of food pathogenic bacteria. It has a stronger effective than normal chemical antisept, which is a valid symbol for lactic acid bacteria against fungus. PLA has a broad inhibitory spectra, high stability and good solubility, is a novel natural bio-antisept. The research of this paper mainly focused on the fed-batch fermentation of PLA production with pH control in 5 L fermentor. Use traditional microbial fermentation and resting cell conversion methods to produce PLA with the substrate phenylpruvic acid(PPA). The operation is convenient, the target product is certain. Both methods have their own merits, which has a practical significance to the industrialization.First of all, based on the shaking flask fermentation, we did the research on the 5 L fermentor technics, the results showed that the bacterium growth and PLA production were synchronous, the ration of coupling was 80.4 %. Based on the trait of PLA production process, we also did the research on the fed-batch fermentation with pH control, the optimized technics was 3 % seed content, shaking speed was 100 r/min, and we used 10 mol/L NaOH to stabilize the fermentation pH around 6.0. From 12 h to 32 h, every 2 hours added 48mL glucose(500 g/L) and 120 mL phenylpruvic acid(100 g/L), both 11 times. From 42 h to 54 h every 6 hours added 72mL glucose(500 g/L) and 180mL PPA(100 g/L), after 72 h, the PLA content can reach 17.38 g/L, the product strength is 0.241 g/L·h, the PPA transformation efficiency is 48.4 %. Compared with batch fermentation, the biomass is 47 % higher, the PLA yield and poduct strength were 5 times and 1.5 times more respectively.Based on the former lab results, we carried out resting cell bioconversion, the enzyme in the centrifuged resting cell acted upon the PPA. After determining the bacterium age of the resting cell at 18 h, through Plackett-Burrman design, prominent factors were selected. Through Box-Behnken design, the optimum combination was obtained by response surface analysis and ridge analysis. Experiment results indicated that when the bioconversion conditions were the resting cell concentration 9.9 %, the substrate concentration 3.72 g/L, bioconversion temperature 33.4℃, the yield of phenyllactic acid is maximum which predicted value is 2.82 g/L and verified value is 2.88 g/L, which tell us that the regress model can fairly forecast PLA content. We also analyzed the chiral structure of PLA in the resting cell bioconversion production. Both kinds of LDH were in the bioconversion process, and the L-LDH activity is more stronger than D-LDH, at last the content ratio of L-PLA to D-LDH is 2.5:1.Using permeabilized Lactobacillus sp. SK007 cells to produce PLA, and the technical process is: the cell treated with ether at 2 % for 2 min at 30℃. The best pH for the permeabilized cell transfer is 5-7, and 30-45℃for bioconversion to produce PLA is ok. The permeabilization can enhance the satabilization of enzyme and pH range; so when the temperature is 40℃, pH=6.0, PPA consistency is 5 g/L, after 4 hours, the PLA content can reach 3.38 g/L. And the batch times of bioconversion is 3-4. Compared with resting cell, permeabilized cell has broad temperature range and little significance between temperature reaction, during the same reaction time 4 h, and 33.3 % higher upon substrate concentration per gram wet cell, and 20 % higher in PLA production than resting cell, which can provide a new thinking and possibility for PLA industrilization.