Abstract
Cloning of aspartase gene (L-aspartate ammonia-lyase) from the natural isolate Escherichia coli VKPM V-7188 in the pLATE31 plasmid composition and its expression under the control of the T7 phage promoter in Escherichia coli BLR (DE3) cells was performed. It was shown that the aspartase content in recombinant cells under optimal conditions is up to 60% of the sum of all dissolved proteins, while the maximal specific activity of cells increases to 700 µmole/mg S/min. Mutant variants carrying an aspartase gene with end sequence deletions and coding proteins with an increased level of aspartase activity were obtained. The developed system for a highly efficient expression of aspartase gene can be used for the selection and quick estimation of catalytic properties of enzymatic mutant forms generated in vitro, as well as for the creation of new, more efficient biocatalists of L-aspartic acid synthesis.
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Abbreviations
- IPTG:
-
isopropyl-β-D-thiogalactoside
- PMM:
-
phosphate minimal media
- OD:
-
optical density
- PAG:
-
polyacrylamide gel
- PCR:
-
polymerase chain reaction
- LB medium:
-
Luria-Bertani medium
- kbp:
-
kilo base pairs
- CCU:
-
center of collective usage of State Research Institute of Genetics and Selection of Industrial Microorganisms
- SDS:
-
sodium dodecyl sulfate
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Original Russian Text © A.D. Novikov, D.D. Derbikov, O.V. Shaposhnikova, T.A. Gubanova, S.V. Kameneva, A.S. Yanenko, 2014, published in Biotekhnologiya, 2014, No. 5, pp. 19–24.
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Novikov, A.D., Derbikov, D.D., Shaposhnikova, O.V. et al. The highly efficient expression of the aspartase gene (L-aspartate ammonia-lyase) in Escherichia coli cells. Appl Biochem Microbiol 51, 751–756 (2015). https://doi.org/10.1134/S0003683815070042
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DOI: https://doi.org/10.1134/S0003683815070042