Engineering pH tolerant mutants of a cyanide dihydratase of Bacillus pumilus C1 and identifying constraints on substrate specificity in nitrilases

摘要

This study generated two cyanide dihydratase (CynD) mutants of Bacilluspumilus C1 with improved activity at higher pH by random mutagenesis. The purpose ofthis study was to create enzyme variants better suited to degrade cyanide under the harshconditions of industrial applications. We employed error-prone PCR to construct alibrary of CynD mutants. A high throughput screening system was developed to screenthe library for improved activity. Two mutants were identified that could degradecyanide at pH10 whereas the wild-type enzyme was inactive at pH9 or higher. Themutants each had three amino acid substitutions compared to the wild-type enzyme. Themutants were also more stable than the wild-type enzyme at 42oC. E327G was identifiedas 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 Bacillussp. 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 cyanidedihydratases from B. pumilus C1 and P. stutzeri AK61 but utilizes different substrate. This provides a valuable resource to study the substrate specificity determinants ofcyanide degrading enzymes. One deletion mutant and four hybrid proteins wereconstructed based on the alignment information. The constructed proteins were allunable to degrade cyanide.