Noda Institute for Scientific Research, 399 Noda, Noda, Chiba, 278-0037, Japan,
Appl Microbiol Biotechnol. 2013 Oct;97(19):8581-90. doi: 10.1007/s00253-013-4693-4. Epub 2013 Jan 22.
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.
谷氨酰胺酶是一种能够将 L-谷氨酰胺水解为 L-谷氨酸的酶，通过增强鲜味在发酵食品生产中发挥着重要作用。在本研究中，我们通过对已鉴定的谷氨酰胺酶序列进行 BLAST 搜索，在 Aspergillus sojae 基因组中发现了十个谷氨酰胺酶基因。随后，我们构建了谷氨酰胺酶基因敲除突变体。在 Aspergillus sojae 和 Aspergillus oryzae 中，gahB 敲除突变体的谷氨酰胺酶活性降低了约 90%，表明该酶（GahB）占 Aspergillus 物种中谷氨酰胺酶活性的大部分。随后，我们从 AsgahB 过表达转化体中纯化了 GahB 蛋白并对其进行了表征。SDS-PAGE 和凝胶过滤层析分别估计其分子量约为 110 和 259 kDa，表明天然形式的 AsGahB 是二聚体。最适 pH 为 9.0，最适温度为 50°C。底物特异性分析表明，AsGahB 具有肽谷氨酰胺酶-天冬酰胺酶活性，与 AsGahA 相似，但优先选择游离的 L-谷氨酰胺而非游离的 L-天冬酰胺、肽的 C 末端谷氨酰基和天冬酰基残基。
