Process Optimization Of Efficient Enzymatic Conversion For L-Serine And Life Cycle Assessment

L-serine, as a common amino acid, is consist of varied proteins. It is applied in field of medicine, food and cosmetics. L-serine is mainly used for the synthesis of precursors, such as thymine, purine and choline. As a intermediate, it is used for the synthesis of L-dopa, L-tryptophan and L-cysteine. On the base of high catalytic efficiency and specificity, mild reactive conditions, enzymatic method has become a major research direction of L-serine production. However, it has some technical problems with backward detection technology, low conversation rate and yield and severe pollution, to solve these problems, research was carried in this study, and main conclusions were obtained as follows:(1) Based on PITC derivatization, a novel HPLC-ESI-MS method was established for detecting amino acids, the method is of high sensitivity, good reproducibility and selectivity. The mobile phase was 0.05% formic acid aqueous solution (A) and 70:30 (v/v) acetonitrile-water (B). Flow rate was 1 mL min-1.Liquid chromatogram for PITC-amino acids was achieved with an Agilent Eclipse plus C18 column (4.6mm×250mm×5?m) maintained at 30?, UV detection wavelength was 254nm, and injection volume was 10 ?L. Gradient:0 min=90% A and 10% B,12min=70% A and 3% B,20 min=52% A and 48% B,30 min=100% B, complete separation of 15 amino acids in a standard solution (2.5 mmol L-1 for each) derivatized with PITC reagent was achieved within 29 min.(2) Based on the novel HPLC-ESI-MS method, the total ion chromatograms of amino acid standard mixture were analysed. The spectral data of PITC-amino acids are indicative of losses of 17 Da (NH3),18 Da (H2O) and/or 46 Da (CO+H2O) from the parent protonated molecules. Assessment of this method revealed that response curves were linear within the range of 630-20156?mol L-1. LOD and LOQ ranged within 111-816 and 311-2721 pmol ?L-1, respectively. The method was validated with precision values of 0.924%-6.578%(intraday, n=6) and 3.327%-9.845%(interday, n=6 in 3 d).The mean recovery was 82.6-112.0% for amino acid derivatives.(3) The study of regulative mechanism of L-serine synthesis using enzymatic method, a method with rapid reaction rate and high conversion rate was provided for L-serine production. In this study, the strategy of formaldehyde titration was applied (v=1.3 mmol h-1), the activity of serine hydroxymethyltransferase wasnot reduced, glycine was efficiently converted simultaneously, subsequently, the conversion rate of glycine reached 95.6%.(4) The new process route was developed for L-serine preparation.By comparing the static adsorption capacities of four kinds of resins, DAION PA312 resin was selected for the mixed solution separation..The results indicated that the optimal separation conditions: the sample solution pH was 9.0. adsorption time was 20 min, eluting solvent was 0.2 mol L-1phosphate buffer (pH5.0), flow rate was 0.4 mL min-1. The purification method was further determined. Eluent was concentrated to 10:1 (v/v) at 50?, then cooled, aged for 3 h with the addition of 96%(v/v) methanol solution, decolored, crystallized and dried at 80?. Finally, L-serine yield was 75.17%. During the process of separation and purification, mainly pollutants generated were acid and alkali wastewater, abandoned resin, active carbon waste and tiny amount of dust, which were produced in the process of ion-exchange separation, discoloration and drying, respectively.(5) LCA was firstly evaluated for environmental impact, which caused by L-serine synthesis using enzymatic method. Research subjects was 500 t a-1 L-serine production processes, which started form cell disruption using CTAB reagent, ending with L-serine drying, subsquently, model was constructed for life cycle assessment. Based on the emission characteristics of pollution sources, these environmental impacts, such as global warming, acidification, eutrophication, smoke and dust, and solid wastes, was investigated. On the base of data standardization and weighted evaluation, the impact categories affected by L-serine production are eutrophication (3156.447), moke and dust (1464.448), solid wastes (27.776), acidification (13.286) and global warming (2.656), the total environmental impact burden was 4664.653 equivalent unit.