Purification and characterization of a glutaminase enzyme accounting for the majority of glutaminase activity in Aspergillus sojae under solid-state culture

Abstract

Glutaminase, an enzyme that hydrolyzes L-glutamine to L-glutamate, plays an important role in the production of fermented foods by enhancing the umami taste. In this study, we found ten glutaminase genes in the Aspergillus sojae genome by conducting a BLAST search of the characterized glutaminase sequence. We subsequently constructed glutaminase gene disruptants. The glutaminase activity of the gahB disruptant was decreased by approximately 90 % in A. sojae and Aspergillus oryzae, indicating that this enzyme (GahB) accounted for the majority of the glutaminase activity in Aspergillus species. Subsequently, GahB protein was purified from the AsgahB-overexpressing transformant and characterized. The molecular mass was estimated to be approximately 110 and 259 kDa by SDS-PAGE and gel filtration chromatography, respectively, indicating that the native form of AsGahB was a dimer. The optimal pH was 9.0, and the optimal temperature was 50 °C. Analysis of substrate specificity revealed that AsGahB had peptidoglutaminase-asparaginase activity, similar to AsGahA, but preferred free L-glutamine to free L-asparagine, C-terminal glutaminyl, and asparaginyl residues in peptides.

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

“…We found that the glutaminase activity of the gahB disruptant decreased by approximately 90% as compared to the control strain. 16) To determine which glutaminase gene product contributes to glutamate production in soy sauce fermentation, we prepared soy sauces using single disruptants of the 10 glutaminase genes in A. sojae. It is known that the glutamate concentration in soy sauce correlates positively with the glutaminase activity of koji molds.…”

“…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.…”

“…Single gene disruption of 19 enzymes having glutamine deamidase activity was carried out as previously described. 16) Multiple gene disruptions were generated by a loop-out method by repeating gene targeting and pyrG marker recycling (Fig. S1).…”

“…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.…”

“…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. 16) In addition, we constructed several disruptants of the glutaminase genes in A. sojae, and determined that the GahB protein accounted most of the glutaminase activity in koji molds under solid-state culture conditions. 16) Thus, while there are many genes encoding proteins with glutaminase activity in A. oryzae and A. sojae, it remains unclear which of them contribute to glutamate production during soy sauce fermentation.…”