This study generated two cyanide dihydratase (CynD) mutants of Bacillus
pumilus C1 with improved activity at higher pH by random mutagenesis. The purpose of
this study was to create enzyme variants better suited to degrade cyanide under the harsh
conditions of industrial applications. We employed error-prone PCR to construct a
library of CynD mutants. A high throughput screening system was developed to screen
the library for improved activity. Two mutants were identified that could degrade
cyanide at pH10 whereas the wild-type enzyme was inactive at pH9 or higher. The
mutants each had three amino acid substitutions compared to the wild-type enzyme. The
mutants were also more stable than the wild-type enzyme at 42oC. E327G was identified
as one of the key amino acids that are responsible for the improved activity.
The goal of the second project was to convert substrate specificity of the Bacillus
sp. OxB-1 nitrilase to that of a cyanidase by mutagenesis or construction of hybrid genes.
The OxB-1 nitrilase of Bacillus sp. shows a high level of identity with the cyanide
dihydratases from B. pumilus C1 and P. stutzeri AK61 but utilizes different substrate. This provides a valuable resource to study the substrate specificity determinants of
cyanide degrading enzymes. One deletion mutant and four hybrid proteins were
constructed based on the alignment information. The constructed proteins were all
unable to degrade cyanide.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/ETD-TAMU-2765 |
| Date | 15 May 2009 |
| Creators | Wang, Lan |
| Contributors | Benedik, Michael J. |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Thesis, text |
| Format | electronic, application/pdf, born digital |
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