Linked Life Data

12200541

Source:http://linkedlifedata.com/resource/pubmed/id/12200541

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
7
pubmed:dateCreated
2002-8-29
pubmed:abstractText
Benzoylformate decarboxylase (BFD) from Pseudomonas putida was subjected to directed molecular evolution to generate mutants with increased carboligase activity which is a side reaction of the enzyme. After a single round of random mutagenesis mutants were isolated which exhibited a 5-fold increased carboligase activity in aqueous buffer compared to the wild-type enzyme with a high enantiomeric excess of the product (S)-2-hydroxy-1-phenyl-propanone. From the same library, mutants with enhanced carboligase activity in water-miscible organic solvents have been isolated. The selected mutants have been characterized by sequencing, revealing that all mutants carry a mutation at Leu476, which is close to the active site but does not directly interact with the active center. BFD-L476Q has a 5-fold higher carboligase activity than the wild-type enzyme. L476 was subjected to saturation mutagenesis yielding eight different mutants with up to 5-fold increased carboligase activity. Surprisingly, all L476 mutants catalyze the formation of 2-hydroxy-1-phenyl-propanone with significantly higher enantioselectivity than the wild-type enzyme although enantioselectivity was not a selection parameter. Leu476 potentially plays the role of a gatekeeper of the active site of BFD, possibly by controlling the release of the product. The biocatalyst could be significantly improved for its side reaction, the C-C bond formation and for application under conditions that are not optimized in nature.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jul
pubmed:issn
0269-2139
pubmed:author
pubmed:issnType
Print
pubmed:volume
15
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
585-93
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
2002
pubmed:articleTitle
Improving the carboligase activity of benzoylformate decarboxylase from Pseudomonas putida by a combination of directed evolution and site-directed mutagenesis.
pubmed:affiliation
Institut für Enzymtechnologie der Heinrich-Heine-Universität Düsseldorf, im Forschungszentrum Jülich, D-52426 Jülich, Germany. b.ligen@fz-juelich.de
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't