Structure-guided protein engineering of ammonia lyase for efficient synthesis of sterically bulky unnatural amino acids

Bioresources and Bioprocessing

Table 3 Kinetic characterization of the EcMAL variants M6–M8 to substrates 1a and 1b

Enzyme and mutants^a	Substrate	K_m (mM)	K_cat (min⁻¹)	K_cat/K_m (min⁻¹ mM⁻¹)
M8	1a	1.32 ± 0.09	61.76 ± 0.8	46.8
M7	1a	1.25 ± 0.05	46.02 ± 0.4	36.8
M6	1a	1.47 ± 0.05	34.5 ± 0.74	23.5
M8	1b	1.34 ± 0.06	31.48 ± 0.63	23.5
M7	1b	1.96 ± 0.08	21.5 ± 0.38	11.0
M6	1b	1.23 ± 0.03	42.5 ± 0.72	34.6

^aThree parallels were measured for each sample. The reaction mixture contained 500 mM Tris–HCl buffer (pH 8.5), 500 mM NH₄Cl, 20 mM Mg²⁺, 1a: caffeic acid (0.25–10 mM)/1b: cinnamic acid (0.2–10 mM)