Choline oxidase catalyzes the oxidation of choline to glycine betaine via betaine aldehyde in glycine betaine biosynthesis and betaine acts as an osmolyte. Choline oxidase has attracted a great deal of attention because of its wide application in clinical and its potential use in enzymatic betaine production. Therefore, the development of efficient methods for overexpression of choline oxidase will be very valuable. In the present study, the choline oxidase gene was amplified from a newly isolated Gram-positive soil Arthrobacter globiformis strain HYJE003 and was cloned into a pET expression vector. Furthermore, the culture conditions were optimized for overexpression of cloned choline oxidase gene in different hosts for periplasmic expression of the enzyme. Expression host system Rosetta-gami2(DE3)pLysS yielded more cell-free protein and 20 fold higher active enzyme compared to any other reported studies. Terrific Broth media were found to be yielding the highest cell biomass, by applying the optimized culture conditions and purification strategy 20,902 U of choline oxidase was produced with a specific activity of 95 U/mg. The optimum pH and temperature for the enzyme activity were found to be 7 and 37 °C, respectively. Finally, we have demonstrated efficient bioconversion of betaine using overexpressed and purified choline oxidase enzyme. The enzymatically produced betaine was estimated by the formation of betaine reineckate and we were able to produce 0.83 molar of betaine from one molar of choline chloride.

胆碱氧化酶在甘氨酸甜菜碱生物合成中通过甜菜碱醛催化胆碱氧化成甘氨酸甜菜碱，甜菜碱充当渗透剂。胆碱氧化酶因其在临床上的广泛应用及其在酶促甜菜碱生产中的潜在用途而引起了广泛关注。因此，开发有效的过表达胆碱氧化酶的方法将是非常有价值的。在本研究中，胆碱氧化酶基因从新分离的革兰氏阳性土壤球状节杆菌菌株HYJE003中扩增，并克隆到 pET 表达载体中。此外，优化培养条件以在不同宿主中过表达克隆的胆碱氧化酶基因以用于酶的周质表达。表达宿主系统与任何其他报道的研究相比， Rosetta-gami2(DE3)pLysS产生了更多的无细胞蛋白和高出 20 倍的活性酶。通过应用优化的培养条件和纯化策略，发现 Terrific Broth 培养基产生了最高的细胞生物量，产生了 20,902 U 的胆碱氧化酶，比活性为 95 U/mg。发现酶活性的最适pH和温度分别为7和37°C。最后，我们证明了使用过表达和纯化的胆碱氧化酶对甜菜碱进行有效的生物转化。酶促产生的甜菜碱是通过甜菜碱再链的形成来估计的，我们能够从一摩尔氯化胆碱中生产出 0.83 摩尔的甜菜碱。