The gene encoding a thermostable l-aminoacylase (LAA) from Deinococcus radiodurans BCRC12827 was isolated and expressed in transgenic rice under the control of a rice actin gene promoter or a seed-specific promoter, Ose705. The recombinant LAA in the transgenic line Ose705:LAA was specifically detected in rice grains, but not in leaves, and its identity was confirmed by a LC/MS/MS assay. Furthermore, was efficiently purified via affinity chromatography using a nickel column. Enzymatic activity of this rice-produced LAA was determined by HPLC and a maximum activity at pH 8.0 and 45 °C in a phosphate buffer supplemented with the divalent metal ion Co²⁺ using NAc-l-HPA as a substrate was obtained, similar to its host counterpart. This rice-produced LAA maintained approximately 50% enzyme activity after 48 h of incubation under 45 °C and maintained approximately 90% activity compared to a freshly prepared sample after being stored in rice seeds for 4 years. The present study indicated that seed-specific protein production in transgenic rice is a good and safe source for mass production of LAA, and this system can be useful for the production of other biomedical proteins as well.

中文翻译：

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热稳定的1-氨基酰基酶在转基因水稻中的表达和鉴定

在水稻肌动蛋白基因启动子或种子特异性启动子Ose705的控制下，分离并编码了来自放射杜鹃球菌BCRC12827的热稳定的1-氨基酰基酶（LAA）的基因，并在转基因水稻中表达。转基因品系Ose705：LAA中的重组LAA在水稻谷粒中特异性检测到，但在叶片中未检测到，其身份已通过LC / MS / MS分析得以确认。此外，通过使用镍柱的亲和色谱法有效地纯化。通过HPLC测定这种大米生产的LAA的酶活性，并使用NAc- 1在补充有二价金属离子Co ²⁺的磷酸盐缓冲液中在pH 8.0和45°C时测定最大活性。获得-HPA作为底物，类似于其宿主对应物。与新鲜制备的样品在水稻种子中保存4年后相比，该稻米生产的LAA在45°C下孵育48小时后保持约50％的酶活性，并保持约90％的活性。本研究表明，转基因水稻中种子特异性蛋白的生产是大量生产LAA的良好且安全的来源，该系统也可用于生产其他生物医学蛋白。