节杆菌BT801基因文库构建及其乙内酰脲酶基因分离与表达

L-乙内酰脲酶产生菌节杆菌 BT801的染色体DNA经Sau3A I 部分酶切后分离30kb左右的片段，与经HpaI和PstI酶切的黏粒载体Pkc505进行连接，将连接产物用包装蛋白包装，转染大肠杆菌DH5α得到10 000多个转化子，构建成节杆菌BT801的基因组文库。通过薄层层析等方法筛选得到了1个阳性克隆，通过亚克隆得到了乙内酰脲酶的完整基因，该基因能分别利用自身的启动子和T5启动子在大肠杆菌中进行表达产生有活性的蛋白。

Construction of Gene Library of Arthrobacter BT801 and Isolation & Expression of Hydantoinase Gene

Hydantoinase can be widely used in enzymic production of various amino acids. In order to obtain the hydantoinase genes in Arthrobacter BT801, its chromatosomal DNA is isolated and partialy digested with Sau3A I to collect fragments of about 30kb. Then, this fragment is inserted into the Hpa I and Pst I site of cosmid pKC505. The genomic library was thus constructed by packing in vitro with lambda phage package protein and transfecting E. coli DH5alpha. A positive transformant was selected from the library using thin layer chromatography and other methods. A DNA fragment containing complete hydantoinase genes was sequenced by sub-cloning into pUC18. The gene can express active protein under control of its own promoter and T5 promoter in E. coli. The isolation of the gene established foundition for research and application of the hydantoinase.