Cloning, Recombinant Expression and Evaluation of Biochemical Properties of Glutaminase from native strain Alteribacillus bidgolensis

Moafi, Saba; Sarikhan, Sajjad; Angaji, Abdolhamid; Ghafouri, Hossein

doi:10.48311/biot.2025.27531

Cloning, Recombinant Expression and Evaluation of Biochemical Properties of Glutaminase from native strain Alteribacillus bidgolensis

Document Type : Original Research

Authors

Saba Moafi ¹

Sajjad Sarikhan ²

Abdolhamid Angaji ¹

Hossein Ghafouri ³

¹ Department of Cell and Molecular Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran

² Molecular Bank, Iranian Biological Resource Center (IBRC), ACECR, Tehran, Iran

³ Department of Biology, Faculty of Science, Guilan University, Rasht, Iran

https://doi.org/10.48311/biot.2025.27531

Abstract

L-glutaminases (L-glutamine amidohydrolase, 3.5.1.2) belong to the superfamily of serine-dependent β-lactamases and penicillin-binding proteins. L-glutaminases have received much attention in the last few decades due to their catalytic ability to deaminate glutamine to glutamic acid and ammonia; a property that has made them valuable in different industrial applications, especially in medicine. Research on glutaminase has progressed during the last four decades, though slowly in comparison to other industrially important enzymes. The relatively high cost of glutaminase is one of the major drawbacks hindering its industrial applications. The current production levels of glutaminase are also insufficient for the required clinical trials to facilitate its medical uses and for other applications.The purpose of this study is the solubilized recombinant expression and functional assay of L-glutaminase from Alteribacillus bidgolensis (P4BT) in an E. coli BL21 (DE3) expression system. In this study, the L-glutaminase gene was successfully cloned with the pET30a expression vector in the E. coli BL21 (DE3) expression system. Solubilized expression was achieved with the aid of the pG-KJE8 vector, which contains molecular chaperones. Ultimately, the specific activity of the purified and dialyzed enzyme was assessed at 40°C and pH 8, yielding an enzymatic activity of 0.53 ± 0.01 U/mg at a substrate concentration of 8 mM. The Km value for L-glutaminase was calculated at 3.10 mM, with a Vmax of 0.62 U/mg.

Keywords

Cloning

Recombinant Expression

L- glutaminase

Alteribacillus bidgolensis

Chaperone

Subjects

Pharmaceutical Biotechnology

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