Table 1.

DNA and amino acid substitutions in evolved acylase mutants (obtained by random mutagenesis and screening for enhanced activity on CephC)

Clone	Base	Base substitution	Position		AA position	PCR conditions	AA substitution
First round (from wild-type VAC)
M1	808	T → A	1	}	270	1	F → M
	810	T → G	3
M2	643	G → T	1	}	215	1	A → Y
	644	C → A	2
	645	G → C	3
M3	1246	G → T	1		416	1	D → Y
M4	1246	G → T	1		416	1	D → Y
	1249	C → T	1	}	417		H → Y
	1251	C → T	3
Second round (from M4 variant)
M5	130	A → G	1		44	2	I → V
	145	G → T	1		49		E → stop
	315	C → T	3		105		Silent
	1246	G → T	1		416		D → Y
	1249	C → T	1	}	417		H → Y
	1251	C → T	3
M6	64	T → C	1		22	2	S → P
	147	A → G	3		49		Silent
	1180	A → C	1		394		T → P
	1246	G → T	1		416		D → Y
	1249	C → T	1	}	417		H → Y
	1251	C → T	3
Third round (from M2 variant)
M7	266	A → C	2		89	2	E → A
	643	G → T	1	}	215
	644	C → A	2				A → Y
	645	G → C	3
M8	643	G → T	1	}	215	2
	644	C → A	2				A → Y
	645	G → C	3
	809	T → C	2		270		F → S

The amino acid numbering refers to the sequence of the starting acylase (Aramori et al. 1991); see also Figure 1 ▶.