Sinsuwan Sornchai, Yongsawatdigul Jirawat, Chumseng Suchintana, Yamabhai Montarop
Food Protein Research Unit, School of Food Technology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, Thailand.
Protein Expr Purif. 2012 May;83(1):52-8. doi: 10.1016/j.pep.2012.03.001. Epub 2012 Mar 9.
Glutaminase or L-glutamine aminohydrolase (EC 3.5.1.2) is an enzyme that catalyzes the formation of glutamic acid and ammonium ion from glutamine. This enzyme functions in cellular metabolism of every organism by supplying nitrogen required for the biosynthesis of a variety of metabolic intermediates, while glutamic acid plays a role in both sensory and nutritional properties of food. So far there have been only a few reports on cloning, expression and characterization of purified glutaminases. Microbial glutaminases are enzymes with emerging potential in both the food and the pharmaceutical industries. In this research a recombinant glutaminase from Bacillus licheniformis (GlsA) was expressed in Escherichia coli, under the control of a ptac promoter. The recombinant enzyme was tagged with decahistidine tag at its C-terminus and could be conveniently purified by one-step immobilized metal affinity chromatography (IMAC) to apparent homogeneity. The enzyme could be induced for efficient expression with IPTG, yielding approximately 26,000 units from 1-l shake flask cultures. The enzyme was stable at 30°C and pH 7.5 for up to 6h, and could be used efficiently to increase glutamic acid content when protein hydrolysates from soy and anchovy were used as substrates. The study demonstrates an efficient expression system for the production and purification of bacterial glutaminase. In addition, its potential application for bioconversion of glutamine to flavor-enhancing glutamic acid has been demonstrated.
谷氨酰胺酶或L-谷氨酰胺氨基水解酶（EC 3.5.1.2）是一种催化谷氨酰胺形成谷氨酸和铵离子的酶。该酶通过提供各种代谢中间体生物合成所需的氮，在每个生物体的细胞代谢中发挥作用，而谷氨酸在食品的感官和营养特性方面都发挥着作用。到目前为止，关于谷氨酰胺酶的克隆、表达和纯化特性的报道很少。微生物谷氨酰胺酶在食品和制药行业都具有新兴潜力。在本研究中，来自地衣芽孢杆菌的重组谷氨酰胺酶（GlsA）在ptac启动子的控制下在大肠杆菌中表达。重组酶在其C末端带有十肽组氨酸标签，可通过一步固定化金属亲和色谱（IMAC）方便地纯化至表观均一性。该酶可用IPTG诱导高效表达，从1升摇瓶培养物中产生约26,000个单位。该酶在30°C和pH 7.5下稳定长达6小时，当以大豆和凤尾鱼的蛋白水解物为底物时，可有效地用于增加谷氨酸含量。该研究证明了一种用于生产和纯化细菌谷氨酰胺酶的高效表达系统。此外，还证明了其在将谷氨酰胺生物转化为增味谷氨酸方面的潜在应用。
