A microplate screening procedure was developed for directed evolution of CsCE.
Mutant G4-C5 was obtained after four rounds of random mutagenesis and screening.
Mutant G4-C5 has a 2.8-fold increase in activity and a 3.0-fold increase in kcat/Km.
Yield of lactulose catalyzed by mutant G4-C5 was 76% with no epilactose detected.