Immobilisation of lactate dehydrogenase on poly(aniline)-poly(acrylate) and poly(aniline)-poly-(vinyl sulphonate) films for use in a lactate biosensor
Immobilisation of lactate dehydrogenase on poly(aniline)-poly(acrylate) and poly(aniline)-poly-(vinyl sulphonate) films for use in a lactate biosensor
The immobilisation of enzymes on an electrode surface, in such a manner that they retain both substrate specificity and high levels of catalytic activity, is of great importance in bioelectrochemistry. This includes areas such as the development of enzyme-catalysed fuel cell electrodes, biosensors and other biotechnological applications. We have investigated the catalytic activity of hexahistidine tagged variants of lactate dehydrogenase (EC 1.1.1.27) from the thermophile Bacillus stearothermophilus both in solution and when immobilised on poly(aniline)-poly(acrylate) (PANi-PAA) or poly(aniline)-poly(vinyl sulphonate) (PANi-PVS) composite films. Both the C- and N-terminally tagged enzymes are readily immobilised on the modified electrode and catalyse the conversion of lactate and NAD+ to pyruvate and NADH. The NADH that is generated can be readily oxidised at the PANi-modified electrode surface.
In solution, the activity of the C-tagged enzyme (LDH-CHis) was some 30% less that of the wild-type under comparable conditions, whereas the N-tagged enzyme was found to possess essentially the same activity as the wild-type. However, when the enzymes were immobilised on PANi-PAA and PANi-PVS the C-tagged enzyme films showed a higher NADH-dependent current than the wild-type LDH whilst the N-tagged enzyme had the highest of the three. In addition, the C-tagged enzyme film appeared more stable than the wild-type LDH-PANi film. A novel immobilisation chemistry of the enzyme is proposed to account for these observations.
enzyme immobilisation, bioanode, biofuel cell, nad(+), nadh, cofactor
recycling, adenine-dinucleotide nadh, site-directed mutagenesis, bacillus-stearothermophilus, polyaniline films, poly(aniline)-coatedelectrodes, affinity precipitation, alkaline-phosphatase, glucose-oxidase, immobilization, specificity
191-200
Halliwell, Catherine M.
6cc766e4-a027-4380-8604-c91a27301774
Simon, Evelyne
890fd27c-8885-4929-872f-0afc8d076526
Toh, Chee-Seng
271ecaf6-fbed-454e-aa92-c5e645ada23c
Bartlett, Philip N.
d99446db-a59d-4f89-96eb-f64b5d8bb075
Cass, Anthony E.G.
6696e860-4ee0-42ea-958f-2cc9ca999e91
25 February 2002
Halliwell, Catherine M.
6cc766e4-a027-4380-8604-c91a27301774
Simon, Evelyne
890fd27c-8885-4929-872f-0afc8d076526
Toh, Chee-Seng
271ecaf6-fbed-454e-aa92-c5e645ada23c
Bartlett, Philip N.
d99446db-a59d-4f89-96eb-f64b5d8bb075
Cass, Anthony E.G.
6696e860-4ee0-42ea-958f-2cc9ca999e91
Halliwell, Catherine M., Simon, Evelyne, Toh, Chee-Seng, Bartlett, Philip N. and Cass, Anthony E.G.
(2002)
Immobilisation of lactate dehydrogenase on poly(aniline)-poly(acrylate) and poly(aniline)-poly-(vinyl sulphonate) films for use in a lactate biosensor.
Analytica Chimica Acta, 453 (2), .
(doi:10.1016/S0003-2670(01)01408-8).
Abstract
The immobilisation of enzymes on an electrode surface, in such a manner that they retain both substrate specificity and high levels of catalytic activity, is of great importance in bioelectrochemistry. This includes areas such as the development of enzyme-catalysed fuel cell electrodes, biosensors and other biotechnological applications. We have investigated the catalytic activity of hexahistidine tagged variants of lactate dehydrogenase (EC 1.1.1.27) from the thermophile Bacillus stearothermophilus both in solution and when immobilised on poly(aniline)-poly(acrylate) (PANi-PAA) or poly(aniline)-poly(vinyl sulphonate) (PANi-PVS) composite films. Both the C- and N-terminally tagged enzymes are readily immobilised on the modified electrode and catalyse the conversion of lactate and NAD+ to pyruvate and NADH. The NADH that is generated can be readily oxidised at the PANi-modified electrode surface.
In solution, the activity of the C-tagged enzyme (LDH-CHis) was some 30% less that of the wild-type under comparable conditions, whereas the N-tagged enzyme was found to possess essentially the same activity as the wild-type. However, when the enzymes were immobilised on PANi-PAA and PANi-PVS the C-tagged enzyme films showed a higher NADH-dependent current than the wild-type LDH whilst the N-tagged enzyme had the highest of the three. In addition, the C-tagged enzyme film appeared more stable than the wild-type LDH-PANi film. A novel immobilisation chemistry of the enzyme is proposed to account for these observations.
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More information
Published date: 25 February 2002
Keywords:
enzyme immobilisation, bioanode, biofuel cell, nad(+), nadh, cofactor
recycling, adenine-dinucleotide nadh, site-directed mutagenesis, bacillus-stearothermophilus, polyaniline films, poly(aniline)-coatedelectrodes, affinity precipitation, alkaline-phosphatase, glucose-oxidase, immobilization, specificity
Identifiers
Local EPrints ID: 19752
URI: http://eprints.soton.ac.uk/id/eprint/19752
ISSN: 0003-2670
PURE UUID: ad2294fd-8c04-41d6-a072-dee638e489bb
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Date deposited: 20 Feb 2006
Last modified: 16 Mar 2024 02:42
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Contributors
Author:
Catherine M. Halliwell
Author:
Evelyne Simon
Author:
Chee-Seng Toh
Author:
Anthony E.G. Cass
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