PubAg

Main content area

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

Author:
Ito, Kotaro, Hanya, Yoshiki, Koyama, Yasuji
Source:
Applied microbiology and biotechnology 2013 v.97 no.19 pp. 8581-8590
ISSN:
0175-7598
Subject:
Aspergillus oryzae, fermented foods, genes, glutaminase, molecular weight, pH, polyacrylamide gel electrophoresis, proteins, size exclusion chromatography, substrate specificity, taste, temperature
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.
Agid:
607823