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.

FIG 1

Differences in the glycosylation or the specific proteolysis and molecular mass of AsGahA between solid-state culture and submerged culture. The soy sauce koji extracts of the AsGahA-overexpressing transformant (A) and purified AsGahA (B) were deglycosylated by treatment with or without PNGase F. Purified AsGahA was reacted with the soy sauce koji extract of A. oryzae RIB40 (wild type) with (C) or without (D) heat treatment (boiling for 10 min) or with a 10-fold dilution of the soy sauce koji extract without heat treatment (E). Incubation with the soy sauce koji extract was carried out at 30°C for 16 h. Proteins were subjected to SDS-PAGE with a gradient gel (10% to 20%), electroblotted onto a PVDF membrane, and immunodetected using antiserum against the purified AsGahA protein. The peptides A and B are indicated by the closed and open triangles, respectively.