(48h) Immobilization of Threonine Aldolase and Its Application in the Synthesis of Chiral ?-Hydroxy-?-Amino Acid

Abstract: Threonine aldolase can synthesize β-hydroxy-α-amino acids from glycine and aldehyde in one step, which are important intermediate with two chiral centers. The free threonine aldolase is difficult to be separated and reused, and its stablity is not satisfied under industrial condition. Enzyme immobilization technology can effectively solve these problems. Based on the evaluation of enzyme activity recovery and reaction batch stability, immobilization methods of threonine aldolase such as adsorption, entrapment, covalent binding and crossing-linking were studied. After extensive screening, the covalent binding using amino resin with further glutaraldehyde cross-linking appeared to be the most promising method. Then we optimized the immobilization conditions such as the amount of carrier, glutaraldehyde concentration, the activation time for carrier, immobilization time, pH, and temperature. After optimizations, the immobilized enzyme activity recovery was up to 119.3%, and the enantiomeric excess (ee) value of products was 98%. After continuous immersion in the reaction solution for 18 days(enzyme activity is measured daily), the enzyme activity remained 82.4%. And the enzyme activity retention basically unchanged after being stored in a refrigerator at 4 ℃for 62 days. Compared with free enzyme, immobilized threonine aldolase has better batch reaction stability, storage stability and stereoselectivity. This work also contributed to the economic flexibility of β-hydroxy-α-amino acids biosynthesis.