PURPOSE: To obtain the subject enzyme having specific characteristics by isolating GL-7ACA acylase from Pseudomonas strain A14 followed by cloning and by transforming host cells with a vector containing a DNA coding the clone, and then culturing the resulting transformant.
CONSTITUTION: A gene coding GL-7ACA acylase is isolated from Pseudomonas strain A14 (FERM BP-3111) and cloned, and host cells (Escherichia coli) is then transformed with a manifestation vector containing a DNA (pCPA14-1) coding the clone. Then the resultant host cells (transformant) are cultured in a medium. A compound of formula I (R1 is acetoxy, H, etc.; R2 is carboxy 1-6C alkanoyl, D-glutamyl, etc.) is then brought into contact with the resulting culture solution, thus obtaining the objective enzyme having the following characteristics: (1) capable of enzymatically transforming glutaryl 7-ACA, adipyl 7-ACA, etc. into 7-aminocephalosporanic acid; (2) made up of α-subunit [28000 dalton in molecular weight (SDS-PAGE)] and β-subunit [61000 dalton in molecular weight (SDS-PAGE)]; and (3) the N-terminated amino acid sequence for the α-subunit being of formula II.
FUKAGAWA MASAO
TSUMURA MANA
ISHITANI YOSUKE
ONO HIROKI
IWAMI MORITA
TAKANORI HITOSHI
