*4.4. Construction of OYE2y Variants by Site-Directed Mutagenesis*

The variants with single or double substitutions were constructed by site-directed mutagenesis according to the QuikChange Mutagenesis Kit. PCR amplification to introduce substitution was performed in 50 μL of standard PCR mixture with 50 ng of template plasmid DNA and 15 pmol each of the appropriate set of primers using the following temperature cycle; 30 s at 95 ◦C, followed by 30 cycles of 95 ◦C for 15 s, appropriate annealing temperature (55~61 ◦C) for 15 s, and 72 ◦C for 80 s, and the final extension of 5 min at 72 ◦C. The plasmid pEASY-E1-*oye2y* was used as template DNA in the single substitution, while the creation of double mutants was based on the generated OYE2y P76C or R330H gene as the template. The primer sets for single or double substitutions were shown in Tables S1 and S2. The amplified PCR fragments were digested with the restriction enzyme *Dpn* I at 37 ◦C for 1 h, and then the digested DNA was directly introduced into *E. coli* strain BL21(DE3). Each constructed plasmid was confirmed by sequencing. Expression and purification of the resulting OYE2y variants were conducted using the same procedure as OYE2y.
