# Purification and biochemical characterization of l-glutaminase from Aspergillus oryzae with potential biotechnological applications in synthesis of l-theanine and as antitumor agent

## TL;DR

This paper describes the purification and characterization of an l-glutaminase from Aspergillus oryzae, showing its potential for food and cancer treatment applications.

## Contribution

The study presents a highly purified l-glutaminase with enhanced thermal stability and antitumor activity, suitable for biotechnological uses.

## Key findings

- The purified enzyme has a molecular weight of 65 kDa and improved thermal resistance with trehalose.
- The enzyme effectively increases l-theanine and glutamic acid production in the presence of NaCl.
- The enzyme shows strong antitumor activity against lung, liver, and breast cancer cells.

## Abstract

Fungal glutaminase is of great importance in different industries fields. Thus, searching for a significantly catalytic l-glutaminase (Glut) with appraising its biochemical properties and biotechnological applications are the main purposes of this work. The Glut from Aspergillus oryzae was purified with a specific activity 258.33 (U/mg of protein), 215-fold and 36.47% yield. The molecular weight of the purified enzyme was 65 kDa. The purified enzyme exhibited remarkably improved resistance toward higher temperature in the presence of an exogenous trehalose. The enzyme had a greater affinity towards l-glutamine, l-cysteine, l-proline and l-lysine than l-valine, and l-glycine. The essentiality of arginine, tryptophan, histidine, and cysteine residues in the catalysis process of l-glutaminase was determined. The application of biocatalyst in the reaction mixture has not only remarkably increased l-theanine concentration but also has enhanced glutamic acid production in the presence of 10% compared with 15% NaCl. The deamidation of water insoluble Zea or rice glutelin was approximately 65% and 83% after 44 h by the enzymatic treatment. The purified Glut displayed remarkable antitumor activities against lung (A549), liver (HepG2) and human breast (MCF-7) carcinoma. Thus, l-Glut from A. oryzae has the potential to be used in food application and in treatment of various cancer cells.

The online version contains supplementary material available at 10.1038/s41598-025-21904-8.