TY - JOUR
T1 - Altering the substrate specificity of cephalosporin acylase by directed evolution of the β-subunit
AU - Otten, Linda G.
AU - Sio, Charles F.
AU - Vrielink, Johanna
AU - Cool, Robbert H.
AU - Quax, Wim J.
PY - 2002
Y1 - 2002
N2 - Using directed evolution, we have selected an adipyl acylase enzyme that can be used for a one-step bioconversion of adipyl-7-aminodesacetoxycephalosporanic acid (adipyl-7-ADCA) to 7-ADCA, an important compound for the synthesis of semisynthetic cephalosporins. The starting point for the directed evolution was the glutaryl acylase from Pseudomonas SY-77. The gene fragment encoding the β-subunit was divided into five overlapping parts that were mutagenized separately using error-prone PCR. Mutants were selected in a leucine-deficient host using adipyl-leucine as the sole leucine source. In total, 24 out of 41 plate-selected mutants were found to have a significantly improved ratio of adipyl-7-ADCA versus glutaryl-7-ACA hydrolysis. Several mutations around the substrate-binding site were isolated, especially in two hot spot positions: residues Phe-375 and Asn-266. Five mutants were further characterized by determination of their Michaelis-Menten parameters. Strikingly, mutant SY-77N266H shows a nearly 10-fold improved catalytic efficiency (kcat/Km) on adipyl-7-ADCA, resulting from a 50% increase in kcat and a 6-fold decrease in Km, without decreasing the catalytic efficiency on glutaryl-7-ACA. In contrast, the improved adipyl/glutaryl activity ratio of mutant SY-77F375L mainly is a consequence of a decreased catalytic efficiency toward glutaryl-7-ACA. These results are discussed in the light of a structural model of SY-77 glutaryl acylase.
AB - Using directed evolution, we have selected an adipyl acylase enzyme that can be used for a one-step bioconversion of adipyl-7-aminodesacetoxycephalosporanic acid (adipyl-7-ADCA) to 7-ADCA, an important compound for the synthesis of semisynthetic cephalosporins. The starting point for the directed evolution was the glutaryl acylase from Pseudomonas SY-77. The gene fragment encoding the β-subunit was divided into five overlapping parts that were mutagenized separately using error-prone PCR. Mutants were selected in a leucine-deficient host using adipyl-leucine as the sole leucine source. In total, 24 out of 41 plate-selected mutants were found to have a significantly improved ratio of adipyl-7-ADCA versus glutaryl-7-ACA hydrolysis. Several mutations around the substrate-binding site were isolated, especially in two hot spot positions: residues Phe-375 and Asn-266. Five mutants were further characterized by determination of their Michaelis-Menten parameters. Strikingly, mutant SY-77N266H shows a nearly 10-fold improved catalytic efficiency (kcat/Km) on adipyl-7-ADCA, resulting from a 50% increase in kcat and a 6-fold decrease in Km, without decreasing the catalytic efficiency on glutaryl-7-ACA. In contrast, the improved adipyl/glutaryl activity ratio of mutant SY-77F375L mainly is a consequence of a decreased catalytic efficiency toward glutaryl-7-ACA. These results are discussed in the light of a structural model of SY-77 glutaryl acylase.
UR - http://www.scopus.com/inward/record.url?scp=0036829913&partnerID=8YFLogxK
U2 - 10.1074/jbc.M208317200
DO - 10.1074/jbc.M208317200
M3 - Article
C2 - 12198140
AN - SCOPUS:0036829913
SN - 0021-9258
VL - 277
SP - 42121
EP - 42127
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 44
ER -