Glutaminase Production by Aspergillus SSF

Abstract

In the production of fermented foods or seasonings from protein such as soy sauce and miso by enzymes of microorganisms, the taste of the product depends on the amount of glutamic acid in the moromi (mash). Therefore, it is important to increase the amount of glutamic acid in the mash to effectively use the raw material proteins. Glutamic acid is considered to be liberated to the mash by two pathways. One is that glutamic acid, which is a constituent of the protein, is liberated from the protein directly. The other is that glutamine, which is also liberated, is hydrolyzed to glutamic acid by the action of glutaminase (Formula 1). Therefore, glutaminase is thought to be an important key enzyme on which the taste of the product depends. Glutaminase reaction Glutamine + H2O→Glutamic acid + NH3 (Formula 1) Glutaminase is widely distributed in life. Recently, the enzyme is being studied in medical and biochemical fields, and studies about the enzyme in mammals and microorganisms has been reported. Not a few glutaminases have been reported to catalyze the r-glutamyltranspeptidase (GGT) reaction. r-Glutamyltranspeptidase (GGT) reaction (Formula 2) r-Glu-X + Y → r-Glu-Y + X (Transpeptidation) r-Glu-X + H2O→Glutamic acid + X (Hydrolysis) X, Y = Ammonia, Amino acids, Peptides, Amines. As for filamentous fungi, a few studies on glutaminase of the koji mold, Aspergillus oryzae or sojae, have been done from the standpoint of taste of fermented foods. However, the GGT activity of the koji glutaminase has not been studied. A. oryzae MA-27-IM was isolated as a high glutaminase producer from a commercial koji seed for soy sauce fermentation, and a wheat bran koji culture of the strain was prepared to investigate the properties of its glutaminase. Both intracellular and extracellular glutaminases were isolated and purified from A. oryzae MA-27-TM. Polyacrylamide gel electrophoresis of each purified preparation gave a single band with identical electrophoretic mobility. The molecular weights of the intracellular and extracellular glutaminases were estimated to be approximately 113, 000. Both preparations hydrolyzed various r-glutamyl compounds, e. g., L-rglutamyl-p-nitroanilide, theanine and glutathione, besides L-glutamine but did not exhibit asparaginase activity. Further investigations of these preparations indicated that these glutaminases possessed almost the same properties, suggesting their similarity. We investigated the r-glutamyltranspeptidation reaction in this preparations in detail. As the concentration of glycylglycine (r-glutamyi acceptor) increased the formation of r-glutamylglycylglycine was enhanced, while the hydrolysis of glutamine was depressed. Several r-glutamylpeptides were enzymatically formed from L-glutamine and L-amino acids. To investigate the role of glutaminase in soy sauce fermentation, soy bean protein was digested with the enzyme preparation (water extract of wheat bran), and r-glutamylserine, r-glutamylglutamic acid and r-glutamylalanine were isolated from an acidic fraction of the soybean protein digested. The addition of the purified glutaminase from A. oryzae caused more production of glutamic acid and r-glutamylpeptide and less production of glutamine and pyroglutamic acid. And GOT activity and r-glutamylpeptide(s) were also detected in a practical soy sauce mash. In this study, the GOT activity and the GOT reaction of the koji mold glutaminase were studied and the function and significance of the glutaminase and GOT reactions in food fermentation were also studied.