Cyanide is a well known toxicant that arises in the environment from both biological and industrial sources. Bacteria have evolved novel coping mechanisms for cyanide and function as principal agents in the biosphere for cyanide recycling. Some bacteria exhibit the unusual ability of growing on cyanide as the sole nitrogen source. One such organism is Pseudomonas fluorescens NCIMB 11764 (Pf11764) which employs a novel oxidative mechanism for detoxifying and assimilating cyanide. A unique complex of enzymes referred to as cyanide oxygenase (CNO) is responsible for this ability converting cyanide to ammonia which is then assimilated. Because one component of the four member CNO complex was previously shown to act on cyanide independent of the other members, its characterization was sought as a means of gaining a better understanding of the overall catalytic mechanism of the complex. Preliminary studies suggested that the enzyme belonged to a subset of nitrilase enzymes known as cyanide dihydratases (CynD), however, a cynD-like gene in Pf11764 could not be detected by PCR. Instead, a separate nitrilase (Nit) linked to cyanide metabolism was detected. The corresponding nit gene was shown to be one of a conserved set of nit genes traced to a unique cluster in bacteria known as Nit1C. To determine whether the previously described CynD enzyme was instead Nit, efforts were undertaken to isolate the enzyme. This was pursued by cloning and expressing the recombinant enzyme and by attempting to isolate the native enzyme. This thesis is concerned with the latter activity and describes the purification of a Nit-like cyanide-degrading nitrilase (NitCC) from Pf11764 to ~95% homogeneity. Purification was greatly facilitated by the discovery that fumaronitrile, as opposed to cyanide, was the preferred substrate for the enzyme (20 versus 1 U/mg protein, respectively). While cyanide was less effective as a substrate, the specificity for cyanide far outweighed that (10,000 fold) of the recombinant enzyme (NitPG) implying that the native NitCC protein purified in this work is different from that of the cloned recombinant. Further evidence of this was provided by molecular studies indicating that the two proteins differ in mass (34.5 and 38 kDa, respectively) and amino acid sequence. In summary, two different Nit enzymes are encoded by Pf11764. While the two share greater than 50% amino acid sequence identity, the results suggest that the native NitCC enzyme purified in this work functions better as a cyanide-degrading nitrilase and is one of four enzyme components comprising CNO required for Pf11764 cyanide assimilation.

Cyanide is acutely toxic to the environment. However, this simple nitrile is used in several industrial applications especially the mining industry. Due to its high affinity to metals, cyanide has been used for years to extract gold and other precious metals from the ore. Cyanide nitrilases are considered for the detoxification of the industrial wastewaters contaminated with cyanide. Their application in cyanide remediation promises cheaper and safer processes compared to chemical detoxification. However, application of these enzymes in industry requires improving their characteristics. The goal of this dissertation is to better understand cyanide nitrilases, in particular the cyanide dihydratase from of Bacillus pumilus and Pseudomonas stutzeri and to improve their activity and stability. The lack of any high resolution structure of these enzymes calls for isolating or screening for mutants showing enhancement in enzyme properties. Described first is a simple and efficient method utilizing in vivo recombination to create recombinant libraries incorporating the products of PCR amplification. This method is useful for generating large pools of randomly mutagenized clones after error-prone PCR mutagenesis. Several parameters were investigated to optimize this technique; length of homology region, vector treatment, induction time and ratio of fragment to vector. Using error-prone PCR for random mutagenesis, several CynDpum mutants were isolated for higher catalysis at pH 7.7. Three point mutations, K93R, D172N and E327K increased the enzyme’s thermostability. The D172N mutation also increased the affinity of the enzyme for its substrate at pH 7.7 suggesting an effect on the active site. However, the A202T mutation located in the dimerization or the A surface rendered the enzyme inactive by destabilizing it. No significant effect on activity at alkaline pH was observed for any of the purified mutants. Lastly, an important region for CynDstut activity was identified in the C-terminus. This same region increased the stability of CynDpum compared to the wild-type enzyme. Also, CynDpum-stut hybrid was found to be highly more stable than CynDpum. This same hybrid exhibited 100% activity at pH9, a pH where the parent enzyme is inactive, and retained 40% of its activity at pH 9.5 making it a true pH tolerant mutant.

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.