The stabilisation and immobilisation of oligomeric enzymes
The stabilisation and immobilisation of oligomeric enzymes
Lactate dehydrogenase (LDH) was immobilised to an agarose support via spacer arms of increasing length (1.2-4.4 nm) by a single covalent linkage per tetramer. Immobilisation of LDH via a short spacer arm (<2-5 nm) resulted in reduced specific activity, thermal stability, and stability in the presence of proteases, and altered apparent Kmvalues. Each of these parameters approaches the value of the corresponding parameter for unmodified or 5,5'-dithiobis-(2-nitro-ibenzoic acid) modified-LDH when the enzyme is immobilised via a longer spacer arm (>2.5 nm). LDH covalently attached to soluble agarose had a considerably higher residual specific activity (88 Units/mg) than agarose-immobilised LDH (-e-1 Unit/mg), but after removal of excess soluble agarose and unconjugated enzyme by affinity chromatography on immobilised N -(6-Aminohexyl)-AMP and immobilised Crotalaria juncealectin respectively, had reduced thermal stability. LDH immobilisedto nylon microspheres had a poor residual activity (<4 Units/mg) and had reduced thermal stability. LDH was modified by amidination and acylation to give derivatives that could be copolymerised into polyacrylamide gels. A marked increase in thermal stability was observed on amidination but destabilisation was observed on acylation. These results are discussed in terms of retention of loss of the positive charge onenzyme lysyl residues. Thermal stabilisation of polyacrylamideimmobilised-LDH was observed only when a large number of linkages exist between the enzyme and the gel. Incorporation of functional groups into the polyacrylamide-LDH copolymer reveals that the enzyme is stabilised by nitrile groups. Charged polyacrylamide copolymers stabilise the enzyme if di- or trivalent anions are present. The effect of various aqueous/organic cosolvent mixtures on polyacrylamide immobilised LDH stability was examined and related to the polarity index of the solvent. The dissociation constant (Kd) for NADH for each enzyme derivative was estimated by the quenching of intrinsic protein fluorescence. The properties of a number of other enzymes immobilised by copolymerisation into polyacrylamide gels after amidination or acylation were investigated. A novel heterobifunctional reagent, N-acryloyl-(S-thionitrobenzoic acid)-2-mercaptoethylamine, was synthesised. LOH, polythiolated by amidination or acylation was immobilised to thiolated-agarose, or, after acryloylation with the novel reagent, was copolymerised into polyacrylamide gel. The properties of the resulting immobilised enzyme derivatives are discussed in terms of the number and spatial organisation of the linkages between the enzyme and the support, and the rigidity of the support.
University of Southampton
1981
Ryde, John Roland
(1981)
The stabilisation and immobilisation of oligomeric enzymes.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Lactate dehydrogenase (LDH) was immobilised to an agarose support via spacer arms of increasing length (1.2-4.4 nm) by a single covalent linkage per tetramer. Immobilisation of LDH via a short spacer arm (<2-5 nm) resulted in reduced specific activity, thermal stability, and stability in the presence of proteases, and altered apparent Kmvalues. Each of these parameters approaches the value of the corresponding parameter for unmodified or 5,5'-dithiobis-(2-nitro-ibenzoic acid) modified-LDH when the enzyme is immobilised via a longer spacer arm (>2.5 nm). LDH covalently attached to soluble agarose had a considerably higher residual specific activity (88 Units/mg) than agarose-immobilised LDH (-e-1 Unit/mg), but after removal of excess soluble agarose and unconjugated enzyme by affinity chromatography on immobilised N -(6-Aminohexyl)-AMP and immobilised Crotalaria juncealectin respectively, had reduced thermal stability. LDH immobilisedto nylon microspheres had a poor residual activity (<4 Units/mg) and had reduced thermal stability. LDH was modified by amidination and acylation to give derivatives that could be copolymerised into polyacrylamide gels. A marked increase in thermal stability was observed on amidination but destabilisation was observed on acylation. These results are discussed in terms of retention of loss of the positive charge onenzyme lysyl residues. Thermal stabilisation of polyacrylamideimmobilised-LDH was observed only when a large number of linkages exist between the enzyme and the gel. Incorporation of functional groups into the polyacrylamide-LDH copolymer reveals that the enzyme is stabilised by nitrile groups. Charged polyacrylamide copolymers stabilise the enzyme if di- or trivalent anions are present. The effect of various aqueous/organic cosolvent mixtures on polyacrylamide immobilised LDH stability was examined and related to the polarity index of the solvent. The dissociation constant (Kd) for NADH for each enzyme derivative was estimated by the quenching of intrinsic protein fluorescence. The properties of a number of other enzymes immobilised by copolymerisation into polyacrylamide gels after amidination or acylation were investigated. A novel heterobifunctional reagent, N-acryloyl-(S-thionitrobenzoic acid)-2-mercaptoethylamine, was synthesised. LOH, polythiolated by amidination or acylation was immobilised to thiolated-agarose, or, after acryloylation with the novel reagent, was copolymerised into polyacrylamide gel. The properties of the resulting immobilised enzyme derivatives are discussed in terms of the number and spatial organisation of the linkages between the enzyme and the support, and the rigidity of the support.
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Published date: 1981
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Local EPrints ID: 459558
URI: http://eprints.soton.ac.uk/id/eprint/459558
PURE UUID: f04ea1b5-447b-4a4d-b1c1-e97ece768b27
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Date deposited: 04 Jul 2022 17:14
Last modified: 04 Jul 2022 17:14
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Author:
John Roland Ryde
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