[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.
Hao
Shu-Feng
SF
Beijing Institute of Biotechnology, Beijing 100071, China.
Zhang
Wei-Cai
WC
Yuan
Hong-Jie
HJ
Wang
Heng-Liang
HL
Huang
Liu-Yu
LY
D000581:Amidohydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D001173:Arthrobacter / Q000235:genetics*; D010582:Bacteriophage lambda / Q000235:genetics; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D011401:Promoter Regions, Genetic / Q000235:genetics
D000581:Amidohydrolases; C020047:dihydropyrimidinase