Engineering and Directed Evolution of a Ca²⁺ Binding Site A-Deficient AprE Mutant Reveal an Essential Contribution of the Loop Leu₇₅–Leu₈₂ to Enzyme Activity

Table 1

Bacterial strains used in this study.

Bacterial strain	Genotype and description	Reference or source
Bacillus subtilis
168	trpC2	W. Nicholson
1A751	eglSΔ102 bglT/bglSΔEV aprE nprE his	BGSC*
PERM222	B. subtilis 1A751 containing pPERM222 (1.2 kbp PCR fragment containing wild-type aprE ORF subcloned in pUSH2	This study
PERM570	B. subtilis 1A751 eglSΔ102 bglT/bglSΔEV aprE nprE his mutM:Tet^𝑟, mutY:Sp^𝑟, sodA:Cm^𝑟 containing pPERM494 (1.2 kbp BamHI fragment from aprE ΔL75–L82 cloned in PUSH2)	This study
PERM505	B. subtilis 1A751 containing pPERM494 (1.2 kbp BamHI fragment from aprE ΔL75–L82 cloned in PUSH2)	This study
PERM658	B. subtilis 1A751 containing pPERM669 (1.2 kbp BamHI fragment from aprE ΔL75–L82 T66M G102D cloned in PUSH2)	This study
PERM200	B. subtilis 1A751 containing pUSH2 (E. coli-B. subtilis shuttle vector for C-terminal His(6)-tagging Cm^𝑟, Kan^𝑟 [12])	This study

*BGSC: Bacillus Genetic Stock Center.