Purification and characterization of a glutaminase enzyme accounting for the majority of glutaminase activity in Aspergillus sojae under solid-state culture
Purification and characterization of a glutaminase enzyme accounting for the majority of glutaminase activity in Aspergillus sojae under solid-state culture
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DOI：
10.1007/s00253-013-4693-4
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发表时间：
2013-10-01
影响因子：
5
通讯作者：
Koyama, Yasuji
中科院分区：
文献类型：
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作者：
Ito, Kotaro;Hanya, Yoshiki;Koyama, Yasuji
谷氨酰胺酶是一种将L-谷氨酰胺水解为L-谷氨酸的酶，在发酵食品的生产中通过增强鲜味而发挥重要作用。在这项研究中，我们发现了10个转氨酶基因在酱油曲霉基因组进行BLAST搜索的特点转氨酶序列。我们随后构建了转氨酶基因破坏物。gahB破坏物的转氨酶活性在A. sojae和曲霉属中，表明该酶（GahB）占曲霉属物种中的大多数转氨酶活性。随后，从AsgahB过表达的酵母中纯化GahB蛋白并表征。通过SDS-PAGE和凝胶过滤色谱法分别估计分子量约为110和259 kDa，表明AsGahB的天然形式是二聚体。最适pH为9.0，最适温度为50 ℃。底物特异性的分析表明，AsGahB的peptidoperaminase-天冬酰胺酶活性，类似于AsGahA，但更喜欢游离L-谷氨酰胺游离L-天冬酰胺，C-末端天冬酰胺，和天冬酰胺残基的肽。
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 A degrees 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.