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Evaluation of Different Glutaryl Acylase Mutants to Improve the Hydolysis of Cephalosporin C in the Absence of Hydrogen Peroxide
Author(s) -
LópezGallego Fernando,
Betancor Lorena,
Sio Charles F.,
Reis Carlos R.,
Jimenez Pol Nadal,
Guisan Jose M.,
Quax Wim. J.,
FernandezLafuente Roberto
Publication year - 2008
Publication title -
advanced synthesis and catalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.541
H-Index - 155
eISSN - 1615-4169
pISSN - 1615-4150
DOI - 10.1002/adsc.200700320
Subject(s) - chemistry , hydrogen peroxide , cephalosporin c , mutant , yield (engineering) , enzyme , catalase , hydrolysis , stereochemistry , biochemistry , combinatorial chemistry , cephalosporin , materials science , metallurgy , gene , antibiotics
2‐Oxoadipoyl‐7‐ACA is an intermediate in the conversion of cephalosporin C (CPC) to 7‐aminocephalosporanic acid (7‐ACA) when using a new route involving D ‐amino acid oxidase, catalase and glutaryl acylase. A key point in the reaction design is to avoid the accumulation of hydrogen peroxide in the reaction medium as the yields of 7‐ACA decrease in the presence of this compound due to its low stability. Looking for an enzyme with improved activity towards 2‐oxoadipoyl‐7‐ACA, different mutants of glutaryl acylase from Pseudomonas SY‐77 with an improved activity towards adipoyl‐7‐ACA were evaluated. The best results on 2‐oxoadipoyl‐7‐ACA hydrolysis were found with the double mutant Y178F+F375H, which showed a K cat increase of 6.5‐fold and a K m decrease of 3‐fold compared to the wild‐type (wt) enzyme. When this enzyme was tested in the tri‐enzymatic system to convert CPC into 7‐ACA, this mutant permitted us to reach more than an 80 % yield of 7‐ACA using a 3‐fold mass excess compared to DAAO; while the wt enzyme gave only a 40 % yield. Therefore, the application of this new mutant to the one‐pot conversion of CPC to 7‐ACA gives very good result in terms of efficiency, yield and rate of the process.