Studies On Extractive Fermentation For L-Phenylalanine Production By Recombinant Escherichia Coli Immobilized With Microcapsules

As one of the eight amino acids essential to human being, L-Phenylalanine (L-Phe) is an important organic compound intermediate, which can be widely used in food industry, medicine, and chemical engineering. It is a major food additive and anticancer medicament intermediate. What is more, it is the raw material of aspartame synthesis, which made it widely demanded in the market.Fermentation by immobilized cells is one of the broadly used ways to produce L-Phenylalanine. In this study, two innovative immobilized systems——SA-CMC/CaCl₂ and NaCS-PDMDAAC microcapsule systems were used to immobilize recombinant E. coli for the extractive fermentation of L-Phe.The extraction of L-phenylalanine using di-(2-ethylhexyl) phosphoric acid (D2EHPA) as extractant and cyclohexane as diluent was studied firstly. The factors that influence the L-Phe extraction process such as the concentration of L-Phe and D2EHP, the pH value and the temperature as well as back-extraction were investigated. The results showed that the distribution coefficient decreased with increasing temperature and depended on the concentration of L-Phe and the initial pH value of aqueous phase. The study of the mechanism of the extraction process indicated that the extraction complex might be composed of one L-Phe molecule and two D2EHPA molecules. The effect of acids on the back-extraction process of L-Phe was proved to be HCl>HNO₃≥H₂SO₄>CH₃COOH, and the back-extraction ratio increased with increasing HCl concentration and temperature.Then the study on growth characteristics of free recombinant E. coli showed that the cells could reach the stationary phase after 12 h of cultivation and glucose was rapidly consumed during this time and completely consumed at 14 h. The yield of L-Phe reached its maximum value at 10 h but decreased afterwards maybe because of the consumption by free cells at stable time. The extractant was harmful to recombinant E. coli, so in later studies direct contact of cells to the extractant should be avoided. Therefore two innovative immobilized system– SA-CMC/CaCl₂ and NaCS-PDMDAAC microcapsule systems were used in the extractive fermentation of L-Phe by recombinant E. coli for they had good biocompatibility.The growth behavior of recombinant E. coli in SA-CMC/CaCl₂ microcapsules prepared by using different concentrations of the components was examined. From the above investigations, The suitable concentrations for the preparation of SA-CMC/CaCl₂ microcapsules was proved to be (g L^-1) CMC 12-14,sodium alginate (SA) 8-10 and CaCl₂ 80-100. By investigation of the disruption rate of microcapsules at different concentrations of preparation, the results showed that SA-CMC/CaCl₂ microcapsules were very unstable under the presence of extractant solvents. The relatively preferable concentrations were ( g L^-1): CMC 12.0, SA 10.0, and CaCl₂ 100.0. The results of the extractive fermentation using recombinant E. coli immobilized in the microcapsules prepared at the concentrations mentioned above showed that the stationary phase of the microencapsulated cells delayed 2 h compared with the suspension cultivation due to the mass transfer problem. However, the biomass was a little higher. In the meanwhile, 1.70 and 10.40 g L^-1 L-Phe could be obtained by adding the extractant at 0 and 14 h of the extractive fermentation, respectively.NaCS-PDMDAAC microcapsule system used for the extractive fermentation was also studied. The suitable concentrations for the preparation of the microcapsules were 3%-4%(v/v) NaCS and 4%-5% PDMDAAC. By investigation of the disruption rate of the microcapsules at different concentrations of preparation, the results showed that NaCS-PDMDAAC microcapsules were very stable and were not influenced by the extractant. Considering the fact of mass transfer, 3% NaCS and 4%PDMDAAC were suitable for the preparation of the microcapsules to immobilize recombinant E. coli. The results showed that because the membrane of the NaCS-PDMDAAC microcapsules were very compact, the stationary phase of the immobilized cells was reached 4 h later than free cell culture. The ultimate biomass was lower and glucose was not completely consumed, and the rest concentration of glucose was 4 g L^-1. Moreover, at final stage, there was no consumption of L-Phe. In this situation, 12.5 and 13.16 g L^-1 L-Phe could be obtained by adding the extractant at 0 and 22 h of the extractive fermentation, respectively.