Study On Production Of L-Phe By Immobilized Cells Of Rhodotorula Glutinis

L-phenylalanine (L-Phe) is a aromatic amino acid of physiological activity, and is one of essential amino acid for human being and animal, it is mainly used for producing functional food additive aspartame. At present, due to the development and utilization of anti-cancer drug and mixed amino acid transfusion domestic; the limitation of Saccharin production lead to a sharp increase of aspartame demand, the market demand of L-Phe is increase correspondingly.In this paper, L-Phe has been produced from trans-cinnamic acid(t-Ca) with Phenylalanine ammonia lyase(PAL) as biocatalysts by immobilized cells with Rhodotorula glutinis. My thesis described the research on the condition of immobilization, partial enzymology properties of immobilized cells, influence of glutaraldehyde and effecter of the modification on the impact of immobilized cells activity, condition of enzyme reaction and semi-continued batch bioreacter producing L-Phe with immobilized cells.Based on different preserve period of ten strains of Rhodotorula glutinis in our laboratory. Rhodotorula glutinis. 06112-8-3 with Higher PAL Enzyme Activity is elected after primary and secondary screenings.In this study, the agar, polyacrylamide, polyvinyl alcohol, calcium alginate was used as carrier materials. By comparing with each other in the enzyme recovery and mechanical strength of the carriers, the polyacrylamide was selected as better carrier for immobilization cells.Firstly, the monomer concentration, cross-linking agent concentration, entrapped amount and encapsulating time were studied by single factor test. Then, according to the result of single factor test, condition of immobilization was optimized by Response Surface Method. In the first optimization step, a Plackett-Burmanl design was used to evaluate the influence of the four relevant factors. The result showed that monomer concentration and entrapped amount were the predominant influencing factors. Secondly the path of steepest ascent was used to approach the optimal region of above two factors, In the third step the monomer concentration and entrapped amount were further optimized using central composite designs and response surface analysis. The optimized Immobilization conditions were as followings: monomer concentration, 16.7%; cross-linking agent concentration, 2.0%;entrapped amount, 43%; encapsulating time, 45min. Under this condition, the PAL enzymatic activity recovery of immobilized cells achieved 63% which was 31% higher than that under the original conditions,one batch bioconversion by free whole cells.Part of enzymology properties of immobilized cells were studied and compared with free cells.The heat stability tendency of the immobilized cell was consistent with free cell. At the temperature of 40°C to be incubated, the PAL was still stable, However, over the temperature of 60℃, it descended rapidly. Maximum reaction rate of immobilized cell was present to between 4hr～8hr, then that was delay as compared with free cells. Enzymic stability of immobilized cells was significantly enhanced. The immobilized cells possessed some of catalytic active after continued reacting 5 batch, and compared with that. Enzymatic activity of free cells decreased significantly when being used at second time.PAL enzymatic activity of immobilized cells could be improved by adding effector(sorbitol:α= 18:1) and modification of glutaraldehyde(0.05%). Combinations of effector and glutaraldehyde showed that the residual enzymatic activity and enzymatic stability of immobilized cells were greatly improved.Through to optimizing of enzyme reaction conditions of immobilized cells, It was determined the optimum conditions: the pH was 11; the concentration of NH₄⁺ , 3.0mol/l; the concentration of t-Ca, 13g/l; the reaction temperature, 30°C.Primary Study on semi-continued batch bioreacter transformed t-Ca to L-Phe with immobilized cells. 4.78g t-Ca was transformed to 5.33g L-Phe in 0.1L Semi-continued batch bioreactor by immobilized cells containing 20g wet cells (harvested from liquid cultural substance in 1L shake flask), which was equal to the yield of L-Phe reached 53.3g/l in a liquid reaction mixture. the yield was 1.8 times higher than 30g/l of one transformation of whole cells bioconversion test. If further technology optimization on the bioconversion parameter, the enzymatic prepared L-Phe of non-grain way still has industrial development value.