Document Type : Original Paper

Authors

1 1 Department of Molecular Medicine, Institute of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Islamic Republic of Iran 2 Department of Biology, Faculty of Sciences, University of Guilan, Rasht, Islamic Republic of Iran

2 1 Department of Molecular Medicine, Institute of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Islamic Republic of Iran 3 Department of Systems Biotechnology, Institute of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Islamic Republic of Iran

3 2 Department of Biology, Faculty of Sciences, University of Guilan, Rasht, Islamic Republic of Iran

4 3 Department of Systems Biotechnology, Institute of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Islamic Republic of Iran

5 1 Department of Molecular Medicine, Institute of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Islamic Republic of Iran

10.22059/jsciences.2023.341587.1007721

Abstract

This study aimed to produce a recombinant version of glucose oxidase enzyme and introduce an economical method for enzyme immobilization. The pGAPZαA expression vector was utilized, which has a continuous expression and does not need an inducer and medium replacement. Enzyme immobilization was accomplished by adding WHWTYYW heptapeptide, which tends to bind to cellulosic and nitrocellulose surfaces, to bind the enzyme to the above-mentioned surfaces. In this study, the glucose oxidase (GOX)-encoding gene from Aspergillus niger ATCC 9029 was amplified by the polymerase chain reaction (PCR) and cloned into the pGAPZαA vector, and pGAPZαA/GOX was obtained as a control construct. On the other hand, a primer containing the heptapeptide sequence was designed, followed by adding the heptapeptide by the PCR technique to the end of the carboxyl in the GOX-encoding gene in the pGAPZαA/GOX construct and obtaining the pGAPZαA/GOX–heptapeptide construct. Both constructs were integrated into the Pichia pastoris genome by electroporation, and enzymatic activity and protein expression of the recombinant enzyme from both constructs underwent investigation. The amount of GOX production in the pGAPZαA/GOX wild-type construct and pGAPZαA/GOX with heptapeptide was estimated at 0.234 and 0.135 g/l, respectively. The stability of both constructs was evaluated on different surfaces and then compared as well. It was revealed that the enzyme with WHWTYYW heptapeptide has an increased affinity to bind in cellulose and nitrocellulose surfaces compared to the wild-type enzyme, while demonstrating no affinity for binding to the polyvinylidene fluoride paper.

Keywords

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