The relationship between structure and thermostability of a nitrile hydratase from Goebacillus pallidus RAPc8
type: CreativeWork
description: Nitrile hydratases (NHases) are very important biocatalysts for the enzymatic conversion of nitriles to industrially important amides such as acrylamide and nicotinamide. An “ideal” NHase should fulfil several essential criteria including, high substrate conversion rates, being able to tolerate high substrate and product concentrations as well as being highly thermostable. The NHase used in the present study was isolated from Geobacillus pallidus RAPc8, a moderate thermophile. The primary aims of this study were to use random mutagenesis to engineer the G. pallidus RAPc8 NHase towards improved thermostability and then to use X-ray crystallography to investigate the molecular mechanism(s) involved in the enhanced thermostability. Two randomly mutated libraries were constructed using MnCl2 mediated errorprone PCR. The PCR reaction was performed using 0.05 mM and 0.10 mM MnCl2 and a biased dNTP concentration. The hydroxamic acid assay was used to screen the randomly mutated libraries for NHase mutants with enhanced thermostability. Six mutants that exhibited thermostability-enhancing mutations were isolated from the randomly mutated libraries. The thermostabilised mutants contained between 3 and 7 nucleotide changes per NHase operon. The wild-type and four thermostabilised mutant NHases (7D, 8C, 9C, 9E) were over-expressed, purified, crystallised and subjected to X-ray crystallography. The resolution of the diffraction data for the all the mutant NHases were better than the 2.4Å previously obtained for the wild-type G. pallidus NHase. The best quality data was collected for mutant 9E, which diffracted to a resolution of 1.15Å. The high quality crystal structures allowed each thermostability-enhancing mutation to be viewed in detail. As most of the NHase mutants contained multiple mutations, the crystal structures were important in correlating the observed thermostabilisation with the structural effect of the mutations. Analysis of the X-ray crystal structures illustrat…
abstract: Nitrile hydratases (NHases) are very important biocatalysts for the enzymatic conversion of nitriles to industrially important amides such as acrylamide and nicotinamide. An “ideal” NHase should fulfil several essential criteria including, high substrate conversion rates, being able to tolerate high substrate and product concentrations as well as being highly thermostable. The NHase used in the present study was isolated from Geobacillus pallidus RAPc8, a moderate thermophile. The primary aims of this study were to use random mutagenesis to engineer the G. pallidus RAPc8 NHase towards improved thermostability and then to use X-ray crystallography to investigate the molecular mechanism(s) involved in the enhanced thermostability. Two randomly mutated libraries were constructed using MnCl2 mediated errorprone PCR. The PCR reaction was performed using 0.05 mM and 0.10 mM MnCl2 and a biased dNTP concentration. The hydroxamic acid assay was used to screen the randomly mutated libraries for NHase mutants with enhanced thermostability. Six mutants that exhibited thermostability-enhancing mutations were isolated from the randomly mutated libraries. The thermostabilised mutants contained between 3 and 7 nucleotide changes per NHase operon. The wild-type and four thermostabilised mutant NHases (7D, 8C, 9C, 9E) were over-expressed, purified, crystallised and subjected to X-ray crystallography. The resolution of the diffraction data for the all the mutant NHases were better than the 2.4Å previously obtained for the wild-type G. pallidus NHase. The best quality data was collected for mutant 9E, which diffracted to a resolution of 1.15Å. The high quality crystal structures allowed each thermostability-enhancing mutation to be viewed in detail. As most of the NHase mutants contained multiple mutations, the crystal structures were important in correlating the observed thermostabilisation with the structural effect of the mutations. Analysis of the X-ray crystal structures illustrat…
author: Van Wyk, Jennifer Caroline
keywords: Nitriles
date published: 2008
date modified: 2026-07-24T06:00:40Z
provider: University of the Western Cape
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of the Western Cape