Highly efficient enzymatic synthesis of Z-aspartame in aqueous medium via in situ product removal

• Z-Aspartame was highly efficient synthesized in low pH aqueous medium.
• High yield of Z-APM was achieved due to “in situ product removal”.
• Rationalized the inhibition by Z-l-Asp at a relatively higher concentration using molecular docking approach.

Z-Aspartame (Z-APM) was obtained using an aqueous reaction at pH 6.0 based on thermodynamic control and in situ product removal. The lower pH reaction medium led to higher solubilities of the two substrates, l-PheOMe and Z-l-Asp and dramatically lowered the solubility of the product. The higher pH stability of the protease PT121 and the mutant Y114S enabled the pH modulation of the reaction medium to shift the thermodynamic equilibrium toward product synthesis. The reaction-separation coupling provided a “driving force” for the enzymatic synthesis and resulted in high yields of 88.5% (CZ-l-Asp = 50 mM, Cl-PheOMe = 500 mM) and 82.2% (CZ-l-Asp = 100 mM, Cl-PheOMe = 500 mM), without further purification for removing protection group, of Z-aspartame. Moreover, we rationalized the inhibition by Z-l-Asp at a relatively higher concentration on the synthesis of Z-APM using a molecular docking approach.

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