Abstract
The gene encoding thermostable L-2-halo acid dehalogenase ofPseudomonas sp. YL was isolated, and its over-expression system was constructed. Gene library was prepared fromSau3AI fragments of total DNA fromPs. sp. YL, pUC118 as a vector andEscherichia 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.
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Abbreviations
- IPTG:
-
isopropyl β-D-thiogalactopyranoside
- KPB:
-
potassium phosphate buffer
- SDS:
-
sodium dodecyl sulfate
- X-gal:
-
5-bromo-4-chloro-3-indolyl-β-D-galactoside
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Liu, JQ., Kurihara, T., Nardi-Dei, V. et al. Overexpression and feasible purification of thermostable L-2-halo acid dehalogenase ofPseudomonas sp. YL. Biodegradation 6, 223–227 (1995). https://doi.org/10.1007/BF00700461
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DOI: https://doi.org/10.1007/BF00700461