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Alteration of the substrate range of haloalkane dehalogenase by site‐directed mutagenesis
Author(s) -
Holloway Paul,
Knoke Kyle L.,
Trevors Jack T.,
Lee Hung
Publication year - 1998
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(19980820)59:4<520::aid-bit16>3.0.co;2-d
Subject(s) - mutagenesis , active site , site directed mutagenesis , chemistry , dehalogenase , biochemistry , enzyme , mutant , wild type , protein engineering , stereochemistry , substrate (aquarium) , alanine , amino acid , biology , gene , ecology
We attempted to expand the range of chlorinated solvents degraded by Xanthobacter autotrophicus GJ10 to include trichloroethylene by the rational modification of the enzyme haloalkane dehalogenase. The amino acids Phe164, Asp170, Phe172 and Trp175 were individually replaced with alanine by site‐directed mutagenesis. All substitutions produced enzymes with lower than wild type activity with 1,2‐dichloroethane. The Phe164Ala and Asp170Ala mutants were 3 and 2 times more active than was the wild type enzyme in dechlorinating 1,6‐dichlorohexane. The Asp170Ala mutant resembled the wild type enzyme in its relative activity against longer chain substrates. No mutant was active with trichloroethylene. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59: 520–523, 1998.