Study On Enzymatic Conversion Of Trans-Cinnamic Acid To ～(15)N-L-Phenylalanine

As one of eight essential amino acids, L-Phenylalanine is used for transfusion and intermediate of anti-cancer drug, and is an imperative component of aspatame, a new kind of dipeptide sweetener. 15N-L-Phe is a necessary tracer for amino acid and protein metabolization and amidogen transfer. Because it is stable, secure and doesn't do harm to the environment, it tends to substitute the radioactive tracer and is chosen by medical scientist and nutritionist, especially is suitable for pregnant and infant. The thesis primarily investigated the producing process of L-Phe and tried to find a route in which the stock is conveniently acceptable and the cost is comparably low, the operation is simple and the quality of product is excellent. This work will be a preceding step for the production of 15N-L-Phe. Based on the Rhodotorula glutinis AS2.102, the thesis showed the research on the composition and proportion of the enzymatic formation medium, the optimization of the enzymatic reaction condition, the improvement and stabilization of PAL(EC4.3.1.5) activity, the affecting factors of L-Phe production, and the screening of the hyperactive PAL strain.By investigating the effects of medium components and the cultivation condition on the PAL activity, the optimum culture medium and cultivation condition were obtained. The optimum medium was listed as follows(%): corn steep liquor 2.0, soybean cake1.0, glucose 0.5, K2HPO40.1,NaCl0.5,L-Phe0.05;the optimum cultivation condition included inoculation 10%, medium volume 50ml/250ml, 30℃,pH5.5,150rpm,cultivation time 21h.Considering the effect of reaction temperature, pH and the concentration of substrate on the L-Phe production, we determined the optimum enzymatic reaction condition. The result was shown as below: trans-cinnamic acid 1.5%, NH4OH 8mol/L, wet cell weight 2.50g, pH10.5, 30℃, reaction time 72h.Treated by ultraviolet ray, DES and structural analogue, a hyperactive PAL strain was obtained whose PAL specific enzymatic activity is 22.1×10-3U/mg, L-Phe productivity is 6.46g/L, 1.85 fold of that of the comparing one. In order to improve the PAL activity and L-Phe productivity, we conducted a series of experiment and the results showed that: as a PAL inducer, 0.05% L-Phe can increase PAL activity by 18.2%; 0.005% cetylpyridinium chloride can increase PAL activity by 12.4% as a permeabilizing agent; The appropriate cell loading was 3.6(w/w); Adding trans-cinnamic acid to concentration of 0.1mol/L at 3rd,25th,40th hour can increase the concentration of L-Phenylalanine by 9.6%.Under the conditions of our experiments, a maximum concentration of 10.03g/L L-Phenylalanine, 5.87 fold of that of the original one, and a maximum conversion rate of 60% trans-cinnamic acid were obtained.The thesis also showed the research on the separation and purification of the L-Phe, the process and the result are listed as follows: the conversion liquid was centrifuged to remove the cells and the excess ammonia was removed in vacuo from the supernatant. Adjusted to pH2.0 with HCl, the solution deposited the and was filtered to remove the trans-cinnamic acid. The filtrate was passed through a column packed with ion-exchange resin (H+form) to absorb L-Phe. After eluting with water, 0.5mol/L NH4OH elutation, the eluant was decolored through 2% active carbon, and evaporated to dryness in vacuum to give crude crystals of L-Phe. Dissolved in alcohol liquor, and then crystaled twice, the final product was obtained. The elution rate is 96.9%, the yield of L-Phe in the whole process was 70%.