title: 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.
authors: Noda Institute for Scientific Research, 399 Noda, Noda, Chiba, 278-0037, Japan, koito@mail.kikkoman.co.jp.
authors: Kotaro
authors: K
authors: Ito
authors: Yoshiki
authors: Y
authors: Hanya
authors: Yasuji
authors: Y
authors: Koyama
mesh_terms: D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D002850:Chromatography, Gel; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D017353:Gene Deletion; D005972:Glutaminase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D055503:Protein Multimerization; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D004271:DNA, Fungal; D005972:Glutaminase