Overexpression and feasible purification of thermostable L-2-halo acid dehalogenase of Pseudomonas sp. YL.
The gene encoding thermostable L-2-halo acid dehalogenase of Pseudomonas sp. YL was isolated, and its overexpression system was constructed. Gene library was prepared from Sau3AI fragments of total DNA from Ps. sp. YL, pUC118 as a vector and Escherichia coli JM109 as a host. The recombinant cells resistant to bromoacetate, a germicide, were isolated and shown to produce L-2-halo acid dehalogenase. Subsequently, subcloning was carried out with pKK223-3 as a vector, and the length of DNA inserted was reduced to 1.1 kbp. One of the subclones showed very high activity, and the amount of the dehalogenase produced corresponded to about 30% of the soluble protein. From 5 g (wet weight) of cells, 105 mg of dehalogenase was efficiently purified by heat treatment and DEAE-Toyopearl chromatography. This overexpression system provides a large amount of the thermostable enzyme to enable us to study the properties, structure and application of the enzyme.
Liu
J Q
JQ
Laboratory of Microbial Biochemistry, Kyoto University, Japan.
Kurihara
T
T
Nardi-Dei
V
V
Okamura
T
T
Esaki
N
N
Soda
K
K
D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D006358:Hot Temperature; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011549:Pseudomonas / Q000737:chemistry / Q000201:enzymology*
D006867:Hydrolases; C034507:2-haloacid dehalogenase