Effects Of Cell Permeabilization On Phenyllactic Acid Production

Phenyllactic acid(PLA)is a natural broad-spectrum food preservative with the advantages of good stability and strong solubility.In a previous study,recombinant E.coli BL21(p RSFDuet-2ldh-rbs2aad-fdh)was used as a cell catalyst to convert Phenylalanine(PHE)into PLA through resting cell transformation,resulting in a yield of43.8 g/L of PLA.However,15.2 g/L of the intermediate product Phenylpyruvic acid(PPA)still accumulated during the entire PLA synthesis process,and the substrate molar conversion rate was only 78.7%.In order to improve the production of PLA and the molar conversion rate of substrates,first of all,the Physical methods of preparing permeable cells for resting cell transformation were carried out by four Physical methods(drying method,freeze-thaw method,ultrasonic method and Osmotic pressure impact method)permeabilization of cells after the comparison of relevant experimental data,determine that the ultrasonic method should be selected.Subsequently,the transparency conditions of the ultrasonic method are optimized to determine the optimal ultrasonic processing time,duty cycle and power.Then,the conditions for resting cell transformation after cell permeabilization are further optimized under this condition,and the optimal substrate(PHE,Ammonium formate)concentration,p H,temperature,and buffer system are determined.Finally,changes in the concentration of extracellular PHE,PPA and PLA in permeable cells and changes in permeability in resting cells were examined the effect of sonication on the recycling of resting cells was analyzed,and the mechanism of ultrasonic preparation of permeable cells to improve transformation efficiency was analyzed.In addition,through comparison,it was found that the addition of co-substrates(glucose,glycerol and yeast extract)can further increase the yield of PLA.The specific research results are as follows:(1)Permeabilized cells were prepared by drying method,freeze-thaw method,ultrasonic method and osmotic shock method.The recovery rates of bacteria were 82.23%,92.37%,81.77,and 41.36%,respectively.The highest yields of PLA transformed by resting cells were 6.24 g/L,10.75 g/L,12.44 g/L,5.36g/L,combined with the changing trend of different gradient yields of the four methods,ultrasonic treatment was used to prepare permeabilized cells.(2)When the resting cells were obtained by expanding the culture,the ultrasonic permeabilization conditions were optimized,and the optimal ultrasonic permeabilization treatment conditions were determined as the treatment time of 1 min,the duty cycle of 80%,and the power of 30%.The optimal sonication was applied to prepare permeabilized cells.When the amount of cell catalyst in the buffer system was 30 g/L,and the feed was 30 g/L PHE and 5 g/L AMF,the resting cell transformation made the PLA production reach 30 g/L.20.65 g/L.(3)The optimal sonication conditions screened in the previous step were used to prepare permeabilized cells,and the catalytic conditions of permeabilized cells were optimized.The optimal catalytic conditions were determined as follows: the substrate PHE concentration was 60 g/L,and the molar mass of AMF and PHE was ratio was 1.5,PH 7,35°C,PBS buffer.Using this optimal catalytic condition,the yield of PLA reached 52.62 g/L,and the substrate conversion rate was 94.59%.(4)From the changes in the concentrations of PHE,PPA and PLA inside and outside the permeabilized cells,the changes in the permeability of resting cells,and the changes in the stability of permeabilized cells during the catalysis process,the effect of ultrasonic permeabilization on improving the conversion efficiency of PLA was analyzed.The mechanism shows that ultrasonic treatment can promote the conversion of intermediates by improving the permeability of cell membrane,enhancing the ability of cells to absorb and utilize substrates,and enhance the catalytic ability of L-amino acid deaminase(L-AAD),lactate dehydrogenase(LDH)and formate dehydrogenase(FDH),and improve the whole reaction.catalytic efficiency.(5)Comparing the changes in cell stability and PLA production with the addition of cosubstrates glucose,glycerol and yeast extract during the catalysis process and the control group without addition,through comparison,it was found that glucose was the best cosubstrate,and the addition of glucose increased the production of PLA.An increase of 7.1%(from 52.62 g/L to 56.36 g/L).