The present invention provides L-amino acid α-ligase represented by sequence number 1 (SEQ ID NO. 1), which is a mutant protein including substitution of at least 1-3 amino acid residues of amino acid sequence of protein YwfE, with the substitution being at least one of an asparagine (N) residue of the 108th place from the N terminal being substituted with at least one of an alanine (A) residue, a glutamic acid (E) residue and a glutamine (Q) residue, an isoleucine (I) residue of the 112th place from the N terminal being substituted with a valine (V) residue, and a histidine (H) residue of the 378th place from the N terminal being substituted with at least one of a lysine (K) residue or an arginine (R) residue.