A Paenibacillus sp. dextranase mutant pool with improved thermostability and activity.
Random mutagenesis was used to create a library of chimeric dextranase (dex1) genes. A plate-screening protocol was developed with improved thermostability as a selection criterion. The mutant library was screened for active dextranase variants by observing clearing zones on dextran-blue agar plates at 50 degrees C after exposure to 68 degrees C for 2 h, a temperature regime at which wild-type activity was abolished. A number of potentially improved variants were identified by this strategy, five of which were further characterised. DNA sequencing revealed ten nucleotide substitutions, ranging from one to four per variant. Thermal inactivation studies showed reduced (2.9-fold) thermostability for one variant and similar thermostability for a second variant, but confirmed improved thermostability for three mutants with 2.3- (28.9 min) to 6.9-fold (86.6 min) increases in half-lives at 62 degrees C compared to that of the wild-type enzyme (12.6 min). Using a 10-min assay, apparent temperature optima of the variants were similar to that of the wild type (T (opt) 60 degrees C). However, one of these variants had increased enzyme activity. Therefore, the first-generation dextranase mutant pool obtained in this study has sufficient molecular diversity for further improvements in both thermostability and activity through recombination (gene shuffling).
Hild
Erika
E
Department of Chemistry & Biomolecular Sciences, Macquarie University, Sydney, New South Wales, 2109, Australia.
Brumbley
Stevens M
SM
O'Shea
Michael G
MG
Nevalainen
Helena
H
Bergquist
Peter L
PL
D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D003910:Dextranase / Q000235:genetics* / Q000502:physiology; D019020:Directed Molecular Evolution; D004795:Enzyme Stability / Q000235:genetics; D006358:Hot Temperature
D003910:Dextranase