Performance characterization of recombinant l-glutamate oxidase in a micro GOT/GPT sensing system

Sanjay Upadhyay, Naoto Ohgami, Hitoshi Kusakabe, Hiroshi Mizuno, Jiro Arima, Takashi Tamura, Kenji Inagaki, Hiroaki Suzuki

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Large-scale production of l-glutamate oxidase (l-GlOx) from Streptomyces sp. X-119-6 was conducted with the use of an Escherichia coli expression system. The active α2β2γ2-subunit structure of the enzyme was obtained by proteolysis of a precursor form using metalloendopeptidase. The performance of this recombinant enzyme was tested in an amperometric sensing system to determine the l-glutamate concentration. To this end, a thin-film, three-electrode system was formed. Hydrogen peroxide produced by an enzymatic reaction was detected by using a platinum working electrode. The sensitivity of the l-glutamate sensor was 220 nA/mM, and the lower detection limit was 3 μM (S/N = 3). Up to 800 μM, a linear relationship was observed between the output current and the l-glutamate concentration. Furthermore, we used a microanalysis system to determine the activities of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) and, ultimately, to test whether the recombinant enzyme can additionally be used to analyze enzyme activities. A polydimethylsiloxane (PDMS) flow channel with a mixing compartment was also used. The substrate solution and the sample solution for either GOT or GPT were mixed as they were being removed from the flow channel. As time elapsed, the l-glutamate concentration in the mixed solution increased because of the enzymatic reaction of GOT or GPT; consequently, a constant increase in current was observed. The relation between the slope of the response curve and the enzyme activity was linear up to 127 U/l for GOT and to 88 U/l for GPT. The results of this series of experiments demonstrate that recombinant l-GlOx can be used to construct micro sensors and microanalysis systems.

Original languageEnglish
Pages (from-to)570-576
Number of pages7
JournalSensors and Actuators, B: Chemical
Volume119
Issue number2
DOIs
Publication statusPublished - Dec 7 2006

Fingerprint

glutamates
oxidase
Aspartate Aminotransferases
Alanine Transaminase
Glutamic Acid
Oxidoreductases
Enzymes
Microanalysis
Enzyme activity
Channel flow
Proteolysis
enzymes
enzyme activity
Electrodes
Sensors
channel flow
Polydimethylsiloxane
microanalysis
Hydrogen peroxide
Escherichia coli

Keywords

  • Enzyme activity
  • Flow channel
  • GOT
  • GPT
  • l-Glutamate oxidase

ASJC Scopus subject areas

  • Analytical Chemistry
  • Electrochemistry
  • Electrical and Electronic Engineering

Cite this

Performance characterization of recombinant l-glutamate oxidase in a micro GOT/GPT sensing system. / Upadhyay, Sanjay; Ohgami, Naoto; Kusakabe, Hitoshi; Mizuno, Hiroshi; Arima, Jiro; Tamura, Takashi; Inagaki, Kenji; Suzuki, Hiroaki.

In: Sensors and Actuators, B: Chemical, Vol. 119, No. 2, 07.12.2006, p. 570-576.

Research output: Contribution to journalArticle

Upadhyay, Sanjay ; Ohgami, Naoto ; Kusakabe, Hitoshi ; Mizuno, Hiroshi ; Arima, Jiro ; Tamura, Takashi ; Inagaki, Kenji ; Suzuki, Hiroaki. / Performance characterization of recombinant l-glutamate oxidase in a micro GOT/GPT sensing system. In: Sensors and Actuators, B: Chemical. 2006 ; Vol. 119, No. 2. pp. 570-576.
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