Recombinant production of hyperthermostable CelB from Pyrococcus furiosus in Lactobacillus sp

• Published in: Applied microbiology and biotechnology Vol. 96; no. 4; pp. 903 - 912
• Main Authors: Böhmer, N., Lutz-Wahl, S., Fischer, L.
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 01.11.2012; Springer; Springer Nature B.V
• Subjects: Acids; Analysis; Archaeal Proteins - chemistry; Archaeal Proteins - genetics; Archaeal Proteins - metabolism; Bacteria; Batch culture; Biomedical and Life Sciences; Bioreactors; Biotechnological Products and Process Engineering; Biotechnology; Cellulase - chemistry; Cellulase - genetics; Cellulase - metabolism; E coli; Enzyme Stability; Enzymes; Food safety; Food science; Gene Expression; Genetic recombination; Hot Temperature; Kinases; Lactobacillus - genetics; Lactobacillus - metabolism; Lactobacillus casei; Life Sciences; Microbial Genetics and Genomics; Microbiology; Peptides; Pheromones; Plasmids; Prebiotics; Proteins; Pyrococcus furiosus - enzymology; Pyrococcus furiosus - genetics; Recombinant proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Safety and security measures; Studies; Yeasts; Recombinant CelB; pSIP; Food grade
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-012-4212-z

Lactic acid bacteria (LAB) are used widespread in the food industry as traditional starters for various fermented foods. For recombinant protein production, LAB would be superior with view from the food safety demands since most of them are Generally Recognized As Safe organisms. We investigated the two pSIP expression systems, pSIP403 and pSIP409 (Sørvig et al. 2005 ), to produce a hyper-thermophilic β-glycosidase (CelB) from Pyrococcus furiosus in Lactobacillus plantarum NC8 and Lactobacillus casei as hosts, respectively. Both lactobacilli harboring the pSIP409-celB vector produced active CelB in batch bioreactor cultivations (MRS medium) while the specific CelB activity of the cell free extract was about 44 % higher with L. plantarum (1,590 ± 90 nkat/mg protein ) than with L. casei (1,070 ± 66 nkat/mg protein ) using p -nitrophenyl-β-galactoside (pNPGal) as the substrate. A fed-batch bioreactor cultivation of L. plantarum NC8 pSIP409-celB resulted in a specific CelB activity of 2,500 ± 120 nkat p NPGal /mg protein after 28 h. A repeated dosage of the inducer spp-IP did not increase the enzyme expression further. As alternative for the cost intensive MRS medium, a basal whey medium with supplements (yeast extract, Tween 80, NH 4 -citrate) was developed. In bioreactor cultivations using this medium, about 556 ± 29 nkat p NPGal /mg protein of CelB activity was achieved. It was shown that both LAB were potential expression hosts for recombinant enzyme production. The pSIP expression system can be applied in L. casei .