Student Number : 0009756Y -
MSc dissertation -
School of Molecular and Cell Biology -
Faculty of Science / A versatile nitrile-degrading bacterium was isolated through enrichment culturing of soil
samples from Johannesburg, South Africa. It was identified as Rhodococcus rhodochrous
and submitted to the ATCC culture collection as strain BAA-870. This organism was
determined to be a potential biocatalyst in that it contains a two enzyme system with strong
nitrile-converting activity comprising nitrile hydratase and amidase. The development of a
suitable assay for measuring the activity of the enzymes of interest was explored. A pHsensitive
indicator-based assay was found to be suitable only for colorimetrically
identifying highly concentrated enzymes with acid-forming activity. An ophthaldialdehyde-
based fluorimetric assay was found to be applicable to conversions of
select compounds, but the assay could not be used to measure the activity of Rhodoccocus
rhodochrous ATCC BAA-870. High performance liquid chromatography was the most
suitable method for reliable and quantitative measurement of nitrile hydrolysis, and is
applicable to monitoring activities of whole-cell and cell-free extracts. Initial analysis of
six compounds, benzonitrile, benzamide, benzoic acid, hydrocinnamonitrile, 3-hydroxy-3-
phenylpropionitrile and 3-hydroxy-3-phenylpropionic acid, was performed by HPLC to
measure linearly the average retention area, amount and absorbance of the compounds up
to 10 mM concentrations. The conversion of the substrates benzonitrile, benzamide and 3-
hydroxy-3-phenylpropionitrile were further analysed with respect to time and enzyme
concentration. Conversion of benzonitrile to benzamide by the nitrile hydratase was rapid
and could be measured in 10 minutes. Conversion of benzamide to benzoic acid by the
amidase was considered the rate-limiting step and could be followed for 90 minutes of the
reaction at the concentrations tested. Conversion of 3-hydroxy-3-phenylpropionitrile was
linearly measured over 20 minutes. Mass spectral analysis was used to confirm, at a
structural level, relatively less volatile reactant compounds with a higher thermal stability,
including benzamide, 3-hydroxy-3-phenylpropionitrile and 3-hydroxy-3-phenylpropionic
acid. Protein concentration studies indicated that activity against benzonitrile was probably
due to a nitrile hydratase with potent activity rather than a concentrated enzyme, since
formation of benzamide from benzonitrile showed first order reaction kinetics at protein
concentrations less than 0.2 mg/ml. Formation of benzoic acid from benzamide was linear
up to 1.3 mg total protein and product formation from 3-hydroxy-3-phenylpropionitrile was linear up to 1.4 mg total protein. Overlapping activities against benzonitrile and 3-
hydroxy-3-phenylpropionitrile indicate that the nitrile hydratase has differing substrate
specificity for the two compounds, with higher activity toward the small aromatic
mononitrile, benzonitrile, than the arylaliphatic b-hydroxy nitrile, 3-hydroxy-3-
phenylpropionitrile. The nitrile-converting activity of Rhodococcus rhodochrous ATCC
BAA-870 would be suitable for biocatalysis as the conversions take place under a wide pH
range, require low concentrations of enzyme and reactions are fast. Separation of nitrileconverting
activities in Rhodococcus rhodochrous ATCC BAA-870 was undertaken using
various chromatography methods to establish a simple, one-step protocol for biocatalytic
enzyme preparations. HPLC was not suited to assaying nitrile-converting activity in
chromatofocusing fractions, and chromatofocusing Ampholyte buffers were found to
interfere with activity measurements. Gel exclusion chromatography of the soluble protein
extract from Rhodococcus rhodochrous ATCC BAA-870 indicated the enzyme/s
responsible for nitrile hydratase activity are high molecular weight proteins ranging from
40 to 700 kDa in size, while the amidase native enzyme is proposed to be roughly 17 to 25
kDa. SDS-PAGE analysis of gel exclusion and ion exchange chromatography fractions
indicated nitrile converting activity in Rhodococcus rhodochrous ATCC BAA-870 is likely
due to multimer-forming enzymes made up of 84, 56, 48 and 21 kDa subunits. It is
postulated that nitrile hydratase is made up of ab and a2b2 tetramers that may form larger
enzyme aggregates. Ion exchange chromatography was used to separate nitrile hydratase
with high activity against benzonitrile and 3-hydroxy-3-phenylpropionitrile from amidase
activity, and showed that an additional, substrate specific nitrile hydratase may exist in the
organism.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/2016 |
| Date | 15 February 2007 |
| Creators | Frederick, Joni |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 4117825 bytes, application/pdf, application/pdf |
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