High-level expression of recombinant beta-galactosidases in Lactobacillus plantarum and Lactobacillus sakei using a Sakacin P-based expression system.
This work presents the cloning and expression of the genes encoding heterodimeric beta-galactosidases from Lactobacillus reuteri L103, Lactobacillus acidophilus R22, Lactobacillus plantarum WCFS1, and Lactobacillus sakei Lb790. These enzymes consist of two subunits of approximately 73 and 35 kDa, which are encoded by two overlapping genes, lacL and lacM, respectively. We have cloned these genes into the lactobacillal expression vectors pSIP403 and pSIP409, which are based on the sakacin P operon of L. sakei ( Sørvig et al. Microbiology 2005, 151, 2439- 2449 ), and expressed them in the host strains L. plantarum WCFS1 and L. sakei Lb790. Results varied considerably, ranging from 2.23 to 61.1 U/mg of beta-galactosidase activity, depending on the origin of the lacLM genes, the host strain, and the expression vector used. Highest expression levels were obtained in a laboratory cultivation of L. plantarum WCFS1 harboring the plasmid pEH3R containing the lacLM gene from L. reuteri L103. These cultivations yielded approximately 23 000 U of beta-galactosidase activity per liter, corresponding to the formation of roughly 100 mg of recombinant protein per liter of fermentation medium, and beta-galactosidase levels amounted to 55% of the total intracellular protein of the host organism. To further verify the suitability of this expression system, recombinant beta-galactosidase from L. reuteri was purified to apparent homogeneity. The properties of the purified enzyme were essentially identical with the properties of purified native beta-galactosidase from L. reuteri L103. The presented results lead the way to efficient overproduction of beta-galactosidase in a food-grade expression system, which is of high interest for applications in food industry.
Division of Food Biotechnology, Department of Food Sciences and Technology, BOKU University of Natural Resources and Applied Life Sciences Vienna, Muthgasse 18, A-1190 Vienna, Austria.
Elisabeth
E
Halbmayr
Geir
G
Mathiesen
Thu-Ha
TH
Nguyen
Thomas
T
Maischberger
Clemens K
CK
Peterbauer
Vincent G H
VG
Eijsink
Dietmar
D
Haltrich
D001430:Bacteriocins / Q000235:genetics*; D003001:Cloning, Molecular; D005285:Fermentation; D015870:Gene Expression; D007778:Lactobacillus / Q000201:enzymology*; D048191:Lactiplantibacillus plantarum / Q000201:enzymology*; D009876:Operon / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism
D001430:Bacteriocins; D011994:Recombinant Proteins; C087217:sakacin P protein, Lactobacillus; D001616:beta-Galactosidase