Fig. 5: Directed evolution based on CnTYR-N201S and enzyme screening data.

a Enzyme screening for the random mutagenized variants based on CnTYR-N201S with 3-F-L-tyrosine 11 and 3-Cl-L-tyrosine 12. Reaction conditions: 11 and 12 (2.5 mM, 1 equiv.), enzyme lysates (400 μgmL⁻¹) and CuSO₄ (5 μM) with 0, 4 equivalents and 10 equivalents of sodium ascorbate 8, respectively. Reactions were performed at 25 °C for 8 h. The black colour indicates the diphenolase activities of the tyrosinases. Note: WT-RsTYR (Rs)¹⁹, CnTYR (Cn), and CnTYR-N201S (N201S) lysates were used as positive controls and cell lysates of an empty plasmid as a negative control (NC). b Product yields and components of TYR reactions with 4 equiv. of 8 using WT-CnTYR and CnTYR variants towards 3-F-L-tyrosine 11: orange bars represent the catechol product 3-F-L-DOPA 43 by HPLC against product standards, and the blue bars represent the over-oxidation products (determined by analytical HPLC with short retention times). c Product yields and components of TYR reactions with 4 equiv. of 8 using WT-CnTYR and CnTYR variants towards 3-Cl-L-tyrosine 12: pink bars represent the catechol product 3-Cl-L-DOPA 44 by HPLC against product standards, and the grey bars represent the over-oxidation products as determined by analytical HPLC. Experiments were performed in triplicates. Error bars indicate the standard error of the triplicate reactions.