Gene Cloning, Purification, and Characterization of a Novel Peptidoglutaminase-Asparaginase from Aspergillus sojae

Abstract

Glutaminase is an enzyme that catalyzes the hydrolysis of L-glutamine to L-glutamate, and it plays an important role in the production of fermented foods by enhancing the umami taste. By using the genome sequence and expressed sequence tag data available for Aspergillus oryzae RIB40, we cloned a novel glutaminase gene (AsgahA) from Aspergillus sojae, which was similar to a previously described gene encoding a salt-tolerant, thermostable glutaminase of Cryptococcus nodaensis (CnGahA). The structural gene was 1,929 bp in length without introns and encoded a glutaminase, AsGahA, which shared 36% identity with CnGahA. The introduction of multiple copies of AsgahA into A. oryzae RIB40 resulted in the overexpression of glutaminase activity. AsGahA was subsequently purified from the overexpressing transformant and characterized. While AsGahA was located at the cell surface in submerged culture, it was secreted extracellularly in solid-state culture. The molecular mass of AsGahA was estimated to be 67 kDa and 135 kDa by SDS-PAGE and gel filtration chromatography, respectively, indicating that the native form of AsGahA was a dimer. The optimal pH of the enzyme was 9.5, and its optimal temperature was 50°C in sodium phosphate buffer (pH 7.0). Analysis of substrate specificity revealed that AsGahA deamidated not only free L-glutamine and L-asparagine but also C-terminal glutaminyl or asparaginyl residues in peptides. Collectively, our results indicate that AsGahA is a novel peptidoglutaminase-asparaginase. Moreover, this is the first report to describe the gene cloning and purification of a peptidoglutaminase-asparaginase. G lutaminase (glutamine amidohydrolase, EC 3.5.1.2; Gah) catalyzes the hydrolytic deamidation of L-glutamine, resulting in the production of L-glutamate and ammonium. Glutaminases have been identified in bacteria, yeasts, fungi, and mammals (2, 7, 8), and they play an important role in nitrogen metabolism, including glutaminolysis. Since mitochondrial glutaminase is increased in several tumor types and is frequently upregulated in MYC-transformed cells (4), it is thought to represent a potential chemotherapeutic target (30). Moreover, bacterial glutaminase from Achromobacter was shown to exert antileukemic effects in patients with acute lymphoblastic leukemia or acute myeloid leukemia in a preliminary clinical trial (24), and it has attracted significant attention from the pharmaceutical industry due to its potential applications as an anticancer agent.In addition, L-glutamate (monosodium glutamate) is an important umami taste factor. Thus, the deamidation of glutamine is a key process in the food industry in order to enhance the umami taste. The deamidation of glutamine in proteins found in food also improves functional properties-such as solubility, viscosity, gelation, fat emulsification, and foaming-by increasing the number of negative charges in the protein (5). Although the deamidation of proteins can be performed either enzymatically or nonenzymatically (i.e., chemically), enzymatic protein modifi...

Identification of the Glutaminase Genes ofAspergillus sojaeInvolved in Glutamate Production during Soy Sauce Fermentation

“…Indeed, in the overexpression strains, GahA and GahB were secreted into the extracellular space under solid-state culture conditions, but they were partially retained on the cell surface. 15,16) GgtA was purified from a submerged culture broth of koji molds, indicating that it was also localized extracellularly. 14) In contrast, Gls was localized to the cytoplasm in an A. sojae strain in which the gls gene was overexpressed under the control of the amylase promoter (data not shown), but Gls might gradually leak from cells burst by salt during soy sauce fermentation.…”

“…The cell wall-bound and total glutaminase activities of the glutaminase gene disruptants were measured as previously described. 15,16) The glutaminase activities of KGd-T124, KGd-Tall10, and KGd-NT124-4 were very low, and were hence measured by increasing the reaction time.…”

“…The pH of the moromi mash was gradually reduced by the production of amino acids and organic acids, and a pure culture of Zygosaccharomyces rouxii was added when the pH reached approximately 5.2. The moromi mash was stored at [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] C for 4-6 months with occasional brief aeration to mix the contents and to stimulate microbial growth, and then stored anaerobically for alcohol fermentation. A submerged culture of koji molds was prepared as previously described.…”

“…Several enzymes with glutaminase activity have been identified in A. oryzae and A. sojae, [12][13][14][15] but their substrate specificities differ. Recently we identified 12 glutaminase genes in the A. oryzae RIB40 genome and 10 in the A. sojae NBRC4239 genome, which were of four types (type I, gah; type II, ggt; type III, gta; and type IV, gls) based on amino acid homology.…”

“…Soy sauce koji prepared with these disruptants was used in the assay. After preparation of the cell wall-bound fraction as described previously, 15) glutaminase activity was measured by the enzymatic method using glutamate oxidase. Control, KuP8; KGd-T1, ÁgahA-ÁgahB-ÁgahD; KGd-T2, ÁggtA-ÁggtC-ÁggtD; KGd-T3, ÁgtaA-ÁgtaB-ÁgtaC; KGd-T4, Ágls; KGd-T12, ÁgahA-ÁgahB-ÁgahD-ÁggtA-ÁggtC-ÁggtD; KGd-T13, ÁgahA-ÁgahB-ÁgahD-ÁgtaA-ÁgtaB-ÁgtaC; KGd-T14, ÁgahA-ÁgahB-ÁgahD-Ágls; KGd-T123, ÁgahA-ÁgahB-ÁgahD-ÁggtA-ÁggtC-ÁggtD-ÁgtaA-ÁgtaB-ÁgtaC; KGd-T124, ÁgahA-ÁgahB-ÁgahD-ÁggtA-ÁggtC-ÁggtD-Ágls; KGdTall10, ÁgahA-ÁgahB-ÁgahD-ÁggtA-ÁggtC-ÁggtD-ÁgtaA-ÁgtaB-ÁgtaC-Ágls.…”