Return to search

Site Directed Mutagenesis Of Dienelactone Hydrolase

The role of individual amino acid residues of the enzyme dienelactone hydrolase was investigated. Using the polymerase chain reaction (PCR), a 1.9 kbp clcD fragment was amplified and subcloned yielding a 821 bp BamHI to EcoRI clcD subclone in the plasmid pUC19. Site-specific mutants of dienelactone hydrolase were created using mismatched oligonucleotides to prime DNA synthesis. Specifically modified proteins from mutated clcD genes (Arg 81 to alanine, Tyr 85 to phenylalanine and Arg 206 to alanine), were encoded by the mutant clones. Enzyme assays showed that dienelactone hydrolase activity of the mutants Arg 81 and Arg 206 was totally abolished. The DLHase enzyme activity of mutant Tyr 85 is greatly decreased by approximately two thirds.

Identiferoai:union.ndltd.org:unt.edu/info:ark/67531/metadc500900
Date12 1900
CreatorsChen, Wei, 1965-
ContributorsShanley, Mark Stephen, Benjamin, Robert C., O'Donovan, Gerard A.
PublisherUniversity of North Texas
Source SetsUniversity of North Texas
LanguageEnglish
Detected LanguageEnglish
TypeThesis or Dissertation
Formatv, 72 leaves: ill., Text
RightsPublic, Chen, Wei, 1965-, Copyright, Copyright is held by the author, unless otherwise noted. All rights reserved.

Page generated in 0.0018 seconds