Heterologous gene expression in Lactococcus lactis; expression of the Azotobacter vinelandii algE6 gene product displaying mannuronan C-5 epimerase activity.
The Azotobacter vinelandii mannuronan C-5 epimerases AlgE1-7 can be used to improve the properties of the commercially important polysaccharide alginate that is widely used in a variety of products, such as food and pharmaceuticals. Since lactic acid bacteria are generally regarded as safe, they are attractive candidates for production of the epimerases. A. vinelandii genes are GC-rich, in contrast to those of lactic acid bacteria, but we show here that significant expression levels of the epimerase AlgE6 can be obtained in Lactococcus lactis using the nisin-controlled expression system. A 1200-fold induction ratio was obtained resulting in an epimerase activity of 23900 dpm mg(-1) h(-1), using a tritiated alginate substrate. The epimerase was detected by Western blotting and nuclear magnetic resonance spectroscopy analysis of its reaction product showed that the enzyme displayed catalytic properties similar to those produced in Escherichia coli.
Laboratory of Microbial Gene Technology, Department of Chemistry and Biotechnology, Agricultural University of Norway, P.O. Box 5051, N-1432, As, Norway. janet.blatny@ikb.nlh.no
Janet M
JM
Blatny
Helga
H
Ertesvåg
Ingolf F
IF
Nes
Svein
S
Valla
D000464:Alginates / Q000378:metabolism; D016948:Azotobacter vinelandii / Q000235:genetics*; D015153:Blotting, Western; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D000464:Alginates; D011994:Recombinant Proteins; D002238:Carbohydrate Epimerases