title: 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.
authors.affiliation: Research and Development Division, Kikkoman Corporation, Noda-City, Japan. koito@mail.kikkoman.co.jp
authors.forename: Kotaro
authors.initials: K
authors.lastname: Ito
authors.forename: Kenichiro
authors.initials: K
authors.lastname: Matsushima
authors.forename: Yasuji
authors.initials: Y
authors.lastname: Koyama
mesh_terms: D000581:Amidohydrolases / Q000235:genetics*; D001215:Asparaginase / Q000235:genetics*; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D020224:Expressed Sequence Tags; D040461:Gene Components; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013696:Temperature
chemical_list: D017931:DNA Primers; D000581:Amidohydrolases; D001215:Asparaginase; C022331:peptidyl-glutaminase