Biochemical characterization of broad-specificity enzymes using multivariate experimental design and a colorimetric microplate assay: characterization of the haloalkane dehalogenase mutants

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Authors

MARVANOVÁ Soňa NAGATA Yuji WIMMEROVÁ Michaela SÝKOROVÁ Jana HYNKOVÁ Kamila DAMBORSKÝ Jiří

Year of publication 2001
Type Article in Periodical
Magazine / Source Journal of Microbiological Methods
MU Faculty or unit

Faculty of Science

Citation
Web http://www.ncbr.chemi.muni.cz/~jiri/ABSTRACTS/jmm01.html
Field Microbiology, virology
Description The pH indicator dye-based colorimetric method and multivariate experimental design were used for the systematic biochemical characterization of the broad-specificity enzymes haloalkane dehalogenases. Halogenated compounds for characterization of the enzymes were selected using Principal Component Analysis. The substrates were characterised by 24 physico-chemical and structural descriptors. Thirty four substrates were selected for testing out of 194 halogenated compounds. Relative activities determined using the optimised colorimetric microplate assay were validated against the catalytic constants determined by gas chromatography. The applicability of the assay was tested with F151L, F154L and F169L mutants of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26.
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