Co-immobilised aspartase and transaminase for high-yield synthesis of l-phenylalanine

• One-pot synthesis of l-phenylalanine catalysed by coupling the enzymes aspartase and transaminase.
• Enzymes were compatible upon reaction and immobilisation conditions.
• Concentration of substrates and enzymes were optimised.
• Co-immobilisation of enzymes allowed the synthesis of high concentrations of Phe.

l-phenylalanine (Phe) was synthesised by coupling the enzymes aspartase (AspB) catalysing the synthesis of l-aspartate from fumarate and NH4Cl and microbial aspartate transaminase (TA) catalysing the transfer of the amino group from l-aspartate to phenylpyruvate. Phe synthesis was studied with enzymes in solution and immobilised separately and together on amino-epoxy Relizyme® support. Immobilisation efficiencies and recovered activities of co-immobilised enzymes were slightly lower than those obtained when immobilised separately. Substrate and enzyme concentrations for the synthesis reactions were optimised as follows: co-immobilised 0.3 U/mL AspB and 2 U/mL TA, 0.15 M fumarate, 0.3 M NH4Cl, 0.1 M phenylpyruvate, 0.1 mM pyridoxal-5′-phosphate (PLP) at pH 7.5 and 37 °C. Total reaction yield of 83% and Phe yield of 95% were obtained. The initial rates of the reactions catalysed by co-immobilised enzymes were similar to those obtained when the reactions were catalysed by free enzymes, indicating negligible diffusional limitations associated to the application of the co-immobilised enzymes.