No description available.

No description available.

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.

Cyanide-containing waste is an increasingly prevalent problem in today's society. There are many applications that utilize cyanide, such as gold mining and electroplating, and these processes produce cyanide waste with varying conditions. Remediation of this waste is necessary to prevent contamination of soils and water. While there are a variety of processes being used, bioremediation is potentially a more cost effective alternative. A variety of fungal species are known to degrade cyanide through the action of cyanide hydratases, a specialized subset of nitrilases which hydrolyze cyanide to formamide. Here I report on previously unknown and uncharacterized nitrilases from Neurospora crassa, Gibberella zeae, and Aspergillus nidulans. Recombinant forms of four cyanide hydratases from N. crassa, A. nidulans, G. zeae, and Gloeocercospora sorghi were prepared after their genes were cloned with N-terminal hexahistidine purification tags, expressed in Escherichia coli and purified using immobilized metal affinity chromatography. These enzymes were compared according to their relative specific activity, pH activity profiles, thermal stability, and ability to degrade cyanide in the presence of high concentrations of copper and silver. Although all four were relatively similar, the N. crassa cyanide hydratase (CHT) has the greatest thermal stability and widest pH range where activity remained above 50%. N. crassa also demonstrated the highest rate of cyanide degradation in the presence of both metals tested. The CHT of A. nidulans and N. crassa have the highest reaction rate of the four fungal nitrilases evaluated in this work. These data help determine optimization conditions for the possible use of these enzymes in the bioremediation of cyanide-containing waste. Similar to known plant pathogenic fungi, in vivo expression of CHT in both N. crassa and A. nidulans were induced by growth in the presence of KCN (potassium cyanide).

No description available.

No description available.

Thesis (B.S.)--University of Missouri, School of Mines and Metallurgy, 1910. / The entire thesis text is included in file. Typescript. Illustrated by authors. Sidney S. Schmidt received a Bachelor of Science in General Science. Russell B. Caples received a Bachelor of Science in Mining Engineering. Both degrees determined from "1874-1999 MSM-UMR Alumni Directory". Title from title screen of thesis/dissertation PDF file (viewed March 17, 2009) Includes bibliographical references (p. 2, 17-18).

Thesis (B.S.)--University of Missouri, School of Mines and Metallurgy, 1915. / The entire thesis text is included in file. Typescript. Illustrated by authors. Title from title screen of thesis/dissertation PDF file (viewed April 8, 2009)

Thesis (B.S.)--University of Missouri, School of Mines and Metallurgy, 1903. / The entire thesis text is included in file. Typescript. Illustrated by authors. "1901" handwritten on title page. 1903 determined to be the year the degrees were granted from "1874-1999 MSM-UMR Alumni Directory" and "The History of Missouri School of Mines and Metallurgy" by Bonita and Clair V. Mann. Raphael C. Alexander determined to be Raphael Curier Alexander and W. H. Gill determined to be William Harris Gill from "Thirty-Third Annual Catalogue of the School of Mines and Metallurgy". Title from title screen of thesis/dissertation PDF file (viewed October 27, 2008)

Thesis--University of Missouri, School of Mines and Metallurgy, 1916. / The entire thesis text is included in file. Typescript. Illustrated by author. Paul R. Cook determined to be Paul Richardson Cook from "1874-1999 MSM-UMR Alumni Directory". Title from title screen of thesis/dissertation PDF file (viewed April 21, 2009)