The objective of this thesis is to establish biocatalytic methods for the synthesis of the amino acids L-aspartate, L-phenylalanine and β−aminobutyrate, as well as the aromatic amines 1-phenylethylamine and 3-amine-1-phenylbutane, by using biocatalysts with transaminase and ammonia-lyase activity immobilized by covalent attachment techniques (for enzymes) and entrapment (for enzymes and cells). Two methods for the synthesis of the essential aromatic amino acid L-phenylalanine (Phe) were established. The first method was carried out by using L-aspartate transaminase (AAT) from porcine heart as biocatalyst. The immobilization of the enzyme AAT on Eupergit® C, MANA-agarose and LentiKats® supports was optimized, and the three immobilized enzymatic derivatives were used for the synthesis of Phe. AAT immobilized on Eupergit® C and LentiKats® allowed improving the stability of the enzyme as well as reaching reaction yields of Phe over 70%. Moreover, a multi-enzymatic one-pot method for the synthesis of Phe was established by coupling of two consecutive enzymatic reactions catalyzed by aspartase (AspB) and transaminase (AT). The compatibility of both enzymes under the reaction conditions (pH 7,5 and 37°C) was shown; and, the optimum concentration of substrates (0,15 M fumarate, 0,3 M NH4Cl and 0,1 M phenylpyruvate) and enzymes (0,3 U of AspB/mL and 2 U of AT/mL) were established. In these conditions, the global reaction yield was 80%. L-aspartate was synthesized by using the enzyme L-aspartate ammonia-lyase or aspartase (AspB) from Bacillus sp. YM55-1. The enzyme was immobilized on Eupergit® C, MANA-agarose and LentiKats® supports. The immobilized biocatalysts were used for the synthesis of highly concentrated Asp (≥ 60 g/L). Furthermore, the immobilized biocatalysts were efficiently reused in 5 cycles of Asp synthesis, maintaining over 90% of activity and reaching over 90% of conversion in all the cases. The synthesis of the aromatic amine 3-amine-phenylbutane (APB) was carried out by means of cells with ω−transaminase (ω−TA) activity as well as the partially purified enzyme ω−TA. A permeabilization method of the cell membranes with cetrimonium bromide (CTAB) was performed. Cells were immobilized in LentiKats® with 100% retention of the catalytic activity. The enzyme ω−TA was immobilized on several supports, and the best results were obtained with Eupergit® CM and LentiKats® supports. The cells (non-permeabilized and permeabilized) and the enzyme immobilized in LentiKats®, allowed reusing the biocatalysts up to 5 and 10 cycles of synthesis of APB, maintaining around 80 and 70% of the initial activity respectively. Finally, it was shown that the mutated aspartase (AspB-C6) from AspB, catalyzes the regioselective amination of crotonic acid to yield β−aminobutyrate. This enzyme was immobilized on Eupergit® C and MANA-agarose supports, according to the immobilization methods established for AspB on the same supports. The immobilization yields were similar; however the retained activities were lower than those obtained for AspB.
|Date of Award||12 Nov 2012|
|Supervisor||Gregorio Alvaro Campos (Director) & Carmen Lopez Diaz (Director)|