Thesis (Ph. D.)--Johns Hopkins University, 1916. / Biographical sketch.

M.Sc. (Biochemistry) / An Acinetobacter sp. was isolated which had the ability to metabolise both organic nitriles and inorganic cyanide salts. The enzyme responsible for the degradation of the nitrile groups, was found to be an extra-cellular complex. This complex was partially purified and was shown to consist of not only a number of protein fractions, but also a definite lipid fraction which was identified as being fatty acids. The entire complex had a molecular weight of about 80 000 Da. The enzyme complex exhibited a high degree of stability in the crude form, but rapidly lost its activity on further purification. The complex had a Km of 0.154 ug/ml and a Vmax of 0.534 ug/ml/min for KCN as substrate. This Km value indicates that the complex has a high affinity for KCN and may be of use in the removal of cyanide at low levels. The temperature optimum was shown to be 20·C and the pH-optimum 6.5. Fatty acids were produced both in the presence and absence of a nitrile substrate, and it is unclear whether carbon from CN degradation can be channeled into fatty acid synthesis by this particular bacterium. This bacterium was found to be very effective in the degradation of nitrile compounds. The removal of cyanide from mine effluents is of particular interest in South Africa today and no effective biological method is currently in use.

No description available.

<p>Geobacillus pallidus RAPc8 is a thermophilic nitrile-degrading isolate, obtained from thermal sediment samples of a New Zealand hot spring. The G. pallidus RAPc8 NHase gene has been cloned and expressed in E. coli. The recombinant NHase exhibits nitrile-degrading activity at 50 &deg / C, capable of degrading branched, linear and cyclic heteroaromatic nitrile substrates. However, no activity was found on homoaromatic nitrile substrates such as benzonitrile. In the present study, high levels of activity on benzonitrile were detected with a double mutant &beta / F52G&beta / F55L. Kinetic analysis on the mutant enzyme showed an 8-fold decrease in KM with benzonitrile (0.3mM) compared to acrylonitrile (2.6mM). Specificity constants (kcat/KM) of 5900 and 450 s-1.mM-1 were obtained for the double mutant on benzonitrile and acrylonitrile respectively. The amino acid residues lining the substrate channel were identified and the geometric dimensions measured. Cavity calculations revealed a 29% increase in volume and a 13% increase in inner surface area for the substrate channel of the double mutant when compared to the wild type. Surface representation of the wild type structure revealed two extended, curved channels, which are accessible to the bulk solvent from two locations in the heterodimer. The removal of the &beta / F52 may have contributed to the presence of a single channel with two opposing openings across the dimers with no internal blockage. Normal Mode Analysis calculations also indicate a higher intrinsic flexibility of the mutant relative tothe wild type enzyme. The increased flexibility within the mutant NHase could have introduced a functionally relevant aromatic substrate recognition conformation</p>

<p>In this study G. pallidus RAPc8 NHase mutants were screened for reduced substrate inhibition compared to the wild type enzyme. Wild type and mutant enzymes were expressed and purified using hydrophobic interaction chromatography. Amidase coupled enzyme stop assays were conducted using 3-cyanopyridine as a substrate, whereas continuous enzyme kinetics were conducted using acrylonitrile as a substrate.</p>

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 164-173).

No description available.

<p>In this study G. pallidus RAPc8 NHase mutants were screened for reduced substrate inhibition compared to the wild type enzyme. Wild type and mutant enzymes were expressed and purified using hydrophobic interaction chromatography. Amidase coupled enzyme stop assays were conducted using 3-cyanopyridine as a substrate, whereas continuous enzyme kinetics were conducted using acrylonitrile as a substrate.</p>

<p>Geobacillus pallidus RAPc8 is a thermophilic nitrile-degrading isolate, obtained from thermal sediment samples of a New Zealand hot spring. The G. pallidus RAPc8 NHase gene has been cloned and expressed in E. coli. The recombinant NHase exhibits nitrile-degrading activity at 50 &deg / C, capable of degrading branched, linear and cyclic heteroaromatic nitrile substrates. However, no activity was found on homoaromatic nitrile substrates such as benzonitrile. In the present study, high levels of activity on benzonitrile were detected with a double mutant &beta / F52G&beta / F55L. Kinetic analysis on the mutant enzyme showed an 8-fold decrease in KM with benzonitrile (0.3mM) compared to acrylonitrile (2.6mM). Specificity constants (kcat/KM) of 5900 and 450 s-1.mM-1 were obtained for the double mutant on benzonitrile and acrylonitrile respectively. The amino acid residues lining the substrate channel were identified and the geometric dimensions measured. Cavity calculations revealed a 29% increase in volume and a 13% increase in inner surface area for the substrate channel of the double mutant when compared to the wild type. Surface representation of the wild type structure revealed two extended, curved channels, which are accessible to the bulk solvent from two locations in the heterodimer. The removal of the &beta / F52 may have contributed to the presence of a single channel with two opposing openings across the dimers with no internal blockage. Normal Mode Analysis calculations also indicate a higher intrinsic flexibility of the mutant relative tothe wild type enzyme. The increased flexibility within the mutant NHase could have introduced a functionally relevant aromatic substrate recognition conformation</p>

This study has resulted in a route to the first known NHZ functionalized xylenebased crown ether, 5-amino-2-methoxy-1,3-xylyl-18-crown-5. The route involves preparing 5-azido-2-methoxy-1,3-xylyl-18-crown-5 from 5-bromo-2-methoxy-1,3-xylyl18-crown-5 by reacting it in turn with n-BuLi and tosyl azide. 5-Amino-2-methoxy-1,3xylyl-l8-crown-5 was obtained by reducing 5-azido-2-methoxy-1,3-xylyl-l8-crown-5 with aqueous sodium borohydride in the presence of a phase transfer agent. The 'H NMR spectrum of the amino derivative showed NMR signals at 6 3.4-3.7 (crown CHZ), S 4.0 (benzylic), S 4.47 (methoxy), and 6 6.58 (aromatic) ppm. The integrated areas were consistent with the formula, and they also suggested the NH2 protons were in the crown CH2 area. The IR (KBr pellet) spectrum showed bands at 3408 cm' and 3364 cm' corresponding to the N-H asymmetric and symmetric stretches, respectively. This study has also provided a new procedure for the preparation of 4-bromo-2,6-bis(bromomethyl) anisole, which was the intermediate for 5-bromo-2-methoxy-1,3-xylyl-18-crown-5. It involved reacting 4-bromophenol in turn with 30 % formaldehyde, dimethylsulfate, and HBr in acetic acid. / Department of Chemistry