Molecular cloning and characterization of a gene encoding glutaminase from Aspergillus oryzae

• Published in: Applied microbiology and biotechnology Vol. 54; no. 1; pp. 59 - 68
• Main Authors: KOIBUCHI, K, KITAMOTO, K, NAGASAKI, H, YUASA, A, KATAOKA, J
• Format: Journal Article
• Language: English
• Published: Berlin Springer 01.07.2000; Springer Nature B.V
• Subjects: Amino Acid Sequence; Amino acids; Aspergillus oryzae; Aspergillus oryzae - enzymology; Aspergillus oryzae - genetics; Bacteria; Bacteriophage lambda - genetics; Base Sequence; Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; Cloning, Molecular; d-Glutamine; DNA, Complementary; DNA, Fungal; Fundamental and applied biological sciences. Psychology; Genetics; Glutaminase - genetics; glutamine; gtaA gene; Ions; Mass spectrometry; Mission oriented research; Molecular Sequence Data; Molecular weight; Proteins; Reverse Transcriptase Polymerase Chain Reaction; Seasonings; Purification; Nucleotide sequence; Enzyme; Industrial application; Soy sauce; Gene overexpression; Glutaminase; Fungi; Aspergillus oryzae; Gene; Hydrolases; Fungi Imperfecti; Thallophyta
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s002530000329

A glutaminase from Aspergillus oryzae was purified and its molecular weight was determined to be 82,091 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Purified glutaminase catalysed the hydrolysis not only of L-glutamine but also of D-glutamine. Both the molecular weight and the substrate specificity of this glutaminase were different from those reported previously [Yano et al. (1998) J Ferment Technol 66: 137-143]. On the basis of its internal amino acid sequences, we have isolated and characterized the glutaminase gene (gtaA) from A. oryzae. The gtaA gene had an open reading frame coding for 690 amino acid residues, including a signal peptide of 20 amino acid residues and a mature protein of 670 amino acid residues. In the 5'-flanking region of the gene, there were three putative CreAp binding sequences and one putative AreAp binding sequence. The gtaA structural gene was introduced into A. oryzae NS4 and a marked increase in activity was detected in comparison with the control strain. The gtaA gene was also isolated from Aspergillus nidulans on the basis of the determined nucleotide sequence of the gtaA gene from A. oryzae.