Linked Life Data

12198140

Source:http://linkedlifedata.com/resource/pubmed/id/12198140

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
44
pubmed:dateCreated
2002-10-28
pubmed:abstractText
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 beta-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-77(N266H) shows a nearly 10-fold improved catalytic efficiency (k(cat)/K(m)) on adipyl-7-ADCA, resulting from a 50% increase in k(cat) and a 6-fold decrease in K(m), without decreasing the catalytic efficiency on glutaryl-7-ACA. In contrast, the improved adipyl/glutaryl activity ratio of mutant SY-77(F375L) 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.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Nov
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
1
pubmed:volume
277
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
42121-7
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
2002
pubmed:articleTitle
Altering the substrate specificity of cephalosporin acylase by directed evolution of the Beta -subunit.
pubmed:affiliation
Department of Pharmaceutical Biology, University Centre for Pharmacy, University of Groningen, Antonius Deusinglaan 1, The Netherlands.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't