Synthesis Of Tryptophan And Non-natural Amino Acids Catalyzed By Recombinant Enzymes

Chiral is one of the basic properties of nature. Most molecules of constituting living organism are asymmetric chiral molecules, which play important physiological functions in vivo. Amino acid is a kind of chiral compound, and is widely used in several industries related to human life and health such as medicine, food, health products, feed and so on. D-amino acids, the synthetic intermediates, are widely used in the preparation of antibiotics, peptide hormone, pesticides and artificial sweeteners. Generally, amino acids can be prepared by fermentation, chemosynthesis or enzymatic synthesis. Compared with chemical synthesis, enzymatic synthesis has many advantages such as high efficiency, environment-friendly and safety. With increased environmental awareness, people have increasingly paid attention to comprehensive utilization of resources, clean production, low-carbon and energy conservation, which have become a global trend of development. Sustainable development, namely with minimal environmental impact, minimal resource consumption and the optimal management model to achieve the most reasonable economic growth, has become consensus on economic and social development fields.1. Recombinant expression of serine hydroxymethyltransferase. The enzyme shows broad substrate specificity, and can optical resolution DL-thero-hydroxy- aminoacid to obtain D-β-hydroxyaminoacid. In the second chapter of the thesis, DL-thero-hydroxyaminoacids were synthesized from benzaldehyde and its derivatives, and steady-state kinetics constants of serine hydroxymethyltransferase were researched. The optimal biocatalytic conditions, pH 7.5, reaction temperature 45℃and substrate concentration 200 mmol/L were established. After biocatalytic resolution, enantiomeric excess of product can reach to 97%. The immobilized cells can be continuously used 10 times, bead activity did not change significantly the first five times they were used, and the average conversion rate during the first five instances was 84.1%. The immobilized cells exhibited favourable operational stability.2. Recombinant expression of tryptophan synthase and tryptophase, the former can synthesize L-tryptophan with serine as substrate, whereas the latter can synthesize L-tryptophan with serine or cysetine as substrate. Tryptophan synthase shows obvious advantages while mixed amino acids mother liquor were used as substrate. In the process of reaction, L-serine contained mixed amino acids mother liquor conversion rate reach 95.1% with a final L-tryptophan concentration of 33.2 g/L. The optimal biocatalytic conditions, pH 8.0, reaction temperature 40℃and 0.04% Tween-80 (10% v/v) were established. There is no significant inhibition on enzymatic activity until ammonium chloride up to 75 g/L, suggesting this enzyme has a highly resistant on salinity and suitable for bioconversion of mixed amino acids mother liquor. Quantitive methods of cysteine and tryptophan in transformation system were established, which utilized Cu2+/xylenol orange and formic acid/hydrochloric acid (1:3 v/v) as color reagent, respectively.3. Recombinant expression of aromatic amino acid transaminase. The enzyme can transfer the ammonia from amino donors to keto acids, and synthesize the corresponding amino acids. In the fourth chapter of this thesis,4-methylsulfonyl phenylpyruvic acid and its derivatives were synthesized. Using keto acids as amino receptors, three aromatic amino acids, three branched-chain amino acids and two acidic amino acids as amino acid donors, research the catalytic activity of aromatic amino acid transaminase. Found that L-tryptophan as amino donor,4-methylsulfonyl phenylpyruvic acid as amino receptor, the enzyme presents maximum activity. The reaction equilibrium conversion rate of amino receptor is 84.4%. The optimal biocatalytic conditions, pH 8.5 and reaction temperature 40℃were established. The method also provides us with an alternative synthesis strategy for other non-natural amino acids, such as L-4-nitrophenylalanine, L-4-methoxy phenylalanine etc.4. Take recombinant aspartase as an example, Plackett-Burman and Box-Behnken designs were applied to optimize the fermentation process of recombinant aspartase.11 related factors were screened out to give four important factors, which were further studied for their interactions. Analytic results were verified by a series of large-scale experiments. Found that peptone of 1.05%(m/v), corn plasm 8.23%(m/v), lactose of 1.12%(m/v) and incubation period of 20.78 h were the optimal conditions. Under these conditions, the maximum predicted activity of aspartase was 3985 U. The maximum activity of aspartase was recorded as 4100.5 U. As compared with the basal culture conditions, the initial reaction rate, molar conversion rate, substrate concentration and bioconversion temperature tolerance also have obviously improved.Based on the developmental trend of amino acids industry and the actual needs of enterprises development, this thesis has made some positive exploration on environment-friendly, resource conservation, low carbon and high efficiency with biocatalysis. Furthermore, with chem-biological synthesis routes, this thesis has synthesized a variety of chiral non-natural amino acids, which has important theoretical significance and potential applications.