Overexpression of a recombinant wildâtype and Hisâtagged Bacillus subtilis glycine oxidase in Escherichia coli
2002
Job, Viviana | Molla, Gianluca | Pilone, Mirella S. | Pollegioni, Loredano
We have cloned the gene coding for the Bacillus subtilis glycine oxidase (GO), a new flavoprotein that oxidizes glycine and sarcosine to the corresponding αâketo acid, ammonia and hydrogen peroxide. By inserting the DNA encoding for GO into the multiple cloning site of the expression vector pT7.7 we produced a recombinant plasmid (pT7âGO). The pT7âGO encodes a fully active fusion protein with six additional residues at the Nâterminus of GO (MARIRA). In BL21(DE3)pLysS Escherichia coli cells, and under optimal isopropyl thioâβâdâgalactoside induction conditions, soluble and active chimeric GO was expressed up to 1.14âUâg−1 of cell (and a fermentation yield of 3.82âU·L−1 of fermentation broth). An Nâterminal Hisâtagged protein (HisGO) was also successfully expressed in E.âcoli as a soluble protein and a fully active holoenzyme. HisGO represents ≈ 3.9% of the total soluble protein content of the cell. The Hisâtagged GO was purified in a single step by nickelâchelate chromatography to a specific activity of 1.06âU·mg−1 protein at 25â°C and with a yield of 98%. The characterization of the purified enzyme showed that GO is a homotetramer of ≈â180âkDa with the spectral properties typical of flavoproteins. GO exhibits good thermal stability, with a Tm of 46â°C after 30âmin incubation; its stability is maximal in the 7.0–8.5 pH range. A comparison of aminoâacid sequence and substrate specificity indicates that GO has similarities to other flavoenzymes acting on primary amines and on dâamino acids.
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